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yeast_paired_end_pubs.txt
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yeast_paired_end_pubs.txt
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accession description experiment_title first_created instrument_model instrument_platform library_layout library_selection run_accession sample_accession sample_alias sample_description sampling_platform scientific_name study_accession study_alias study_title fastq_ftp n_samples has_publication publication Stranded strand_text direction_text library_text kit_text has_doi has_pubmed
SAMN08143838 Illumina HiSeq 2500 sequencing; GSM2879618: Wild-type total RNA-Seq biological replicate 1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2879618: Wild-type total RNA-Seq biological replicate 1; Saccharomyces cerevisiae; RNA-Seq 22/12/18 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR6357070 SAMN08143838 GSM2879618 Wild-type total RNA-Seq biological replicate 1 NA Saccharomyces cerevisiae PRJNA421472 GSE107813 Identification of non-coding transcripts regulated by the transcription factor Rap1 by RNA-Seq analysis ftp.sra.ebi.ac.uk/vol1/fastq/SRR635/000/SRR6357070/SRR6357070_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR635/000/SRR6357070/SRR6357070_2.fastq.gz 18 TRUE "Wu ACK et al., \Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor.\"", Mol Cell, 2018 Dec 20;72(6):942-954.e7""" Stranded (Truseq Stranded) "Normalized reads (y axis) for the Watson (W, blue) and Crick (C, red) strands.;nåÊ= 564 Rap1 binding sites, signals for W and C strands were computed separately.;(E) Similar to (D), using scatterplots to display expression changes for antisense (AS) and sense (S) strand windows relative to the coding gene for Rap1 (nåÊ= 141) and Ume6 (nåÊ= 87) -regulated promoters;(F) Heatmaps showing changes in RNA expression on AS or S strands for data described in (E);TSS-seq signal per million reads and total RNA-seq data are plotted for both strands.;Changes in RNA expression were calculated for nåÊ= 141 promoter Rap1 sitesåÊå±100åÊbp for AS and S strands;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);Each library was sequenced on the HiSeq 2500 or 4000 platform (Illumina) and generated ‰ö_45 million 101åÊbp strand-specific paired-end reads per sample, on average.;First strand cDNA synthesis was performed using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMixPCR Kit (KAPA Biosystems) after RNase H (NEB) and RNase cocktail digestion (Ambion);Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems);Libraries were quantified by Qubit and sequenced on the HiSeq 4000 platform (Illumina), and typically generated ‰ö_39 million 76bp strand-specific single-end reads per sample;Fragment counts within specified windows (e.g.,åÊå± 100åÊbp) around the 564 Rap1 sites (1128 intervals total, Watson and Crick strand alignments were assigned to separate intervals) were obtained using the featureCounts tool from the Subread package (version 1.5.1) (Liao etåÊal., 2014);The parameters used were ‰ÛÏ-O‰ÛÒminOverlap 1‰ÛÒnonSplitOnly‰ÛÒprimary -s 2 -p -B -P -d 0 -D 600 -C.‰Û Windows on separate strands were treated as separate intervals for all strand-specific RNA-seq experiments, and only reads which overlapped with the corresponding strand and interval were counted;Uniquely mapped alignments corresponding to the sense and antisense strands were obtained using SAMtools view (version 1.3.1) by using the flags ‰ÛÏ-q 1 -F 20‰Û and ‰ÛÏ-q 1 -f 16,‰Û respectively (Li etåÊal., 2009);To calculate TPM values for each TSS, TSS-seq counts were obtained by quantifying the abundance of reads with the 1st transcribed 5‰Û_ nucleotide withinåÊå± 75åÊbp of annotated TSSs (Park etåÊal., 2014), on the respective strand" "Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction;We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.;The transcriptionally active coding gene promoters, which often express noncoding transcripts in the antisense direction (Neil etåÊal., 2009, Seila etåÊal., 2008, Xu etåÊal., 2009), are a major source;This process is known as divergent or bidirectional transcription;A large fraction of all noncoding transcripts emanate from divergent or bidirectional gene promoters (Neil etåÊal., 2009, Seila etåÊal., 2008, Xu etåÊal., 2009);Typically, divergent noncoding transcripts initiate within or nearby coding gene promoters, but they do not share the same core promoter as transcripts in the coding direction (Andersson etåÊal., 2015, Duttke etåÊal., 2015, Rhee and Pugh, 2012, Scruggs etåÊal., 2015);We find that depletion of Rap1, but not other transcription factors important for RP expression, causes transcription in the divergent direction;Rap1 represses noncoding transcription typically within 50åÊbp of the Rap1 motif, which is uncoupled from transcription regulation in the protein-coding direction;Thus, a sequence-specific transcription factor controls promoter directionality by repressing transcription in the divergent direction;Our work adds a new layer of regulation to various mechanisms that limit expression of aberrant transcripts and defines how promoter directionality is controlled.;In budding yeast, a large fraction of bidirectional promoters express noncoding transcripts, also known as cryptic unstable transcripts (CUTs) or stable unannotated transcripts (SUTs), in the divergent direction (Neil etåÊal., 2009, Xu etåÊal., 2009);Transcription of divergent CUTs and SUTs typically correlates with nucleosome-depleted regions (NDRs) and promoter activity in the coding gene direction;Expression near the Rap1 binding sites was upregulated in both the sense and antisense direction after Rap1 depletion; however, the largest increase was detected in the antisense direction (FigureåÊ2E);When we clustered for the sense direction signals, we observed that transcripts were upregulated both up- and downstream of the Rap1 site (Sc1 and Sc2);Thus, Rap1 represses transcription near the Rap1 binding sites in the antisense, and to lesser extent, the sense direction.;We integrated Rap1 motifs in a fluorescent reporter construct that harbors a divergent promoter transcribing PPT1 in the coding direction and SUT129 in the noncoding direction (pPS) (Figures 3D and S3A) (Marquardt etåÊal., 2014);The repression of SUT129 by Rap1 was not dependent on transcription regulation in the coding direction because in RAP1-AID (IAA or NT) cells the PPT1 signal matched WT (FigureåÊ3E, right panel);As expected, TSS signals decreased in the sense direction downstream of the Rap1 binding sites in Rap1-depleted cells because coding gene expression was reduced (FigureåÊ4B, sense);Interestingly, sequences directly upstream of the canonical coding transcript TSSs displayed increased TSS signals in the sense direction, suggesting that Rap1 is also important for TSS selection (Challal etåÊal., 2018);Rap1 promotes transcription from the coding direction TSS (black arrows) but represses initiation from the divergent TSS;When Rap1 is absent, transcription in the divergent direction occurs.;Our data demonstrate how a sequence-specific transcription factor can prevent regulatory sequences from producing aberrant transcripts and define a mechanism for providing directionality toward productive transcription.;Several lines of evidence indicate that Rap1 represses divergent noncoding transcription, uncoupled from transcription regulation in the coding direction;Fourth, the Rap1 binding site ectopically represses divergent noncoding transcription without affecting transcription in the protein-coding direction;Conversely, the AD of Rap1, which directs transcription in the protein-coding direction, is not required for repressing divergent transcription;Promoter directionality is shaped by evolution toward protein-coding genes through enrichment of DNA binding protein motifs (Jin etåÊal., 2017);In this context, Rap1 promotes directionality in multiple ways;First, Rap1 recruits cofactors and basal transcription machinery, which promote transcription in the coding direction (Azad and Tomar, 2016, Hu and Li, 2007);Core promoters are intrinsically directional (Duttke etåÊal., 2015), and two independent pre-initiation complexes initiate divergent transcription at mRNA-noncoding RNA pairs in yeast (Rhee and Pugh, 2012);Hence, repressing transcription initiation at the antisense core promoter regulates promoter directionality;Our data indicate that Rap1 restricts RSC to stimulate productive transcription in the protein-coding direction" "Total RNA from yeast was incubated with rDNase (Machery-Nagel) and column purified (Machery-Nagel) prior to sequencing library preparation;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);Each library was sequenced on the HiSeq 2500 or 4000 platform (Illumina) and generated ‰ö_45 million 101åÊbp strand-specific paired-end reads per sample, on average.;To obtain libraries representing the 5‰Û_ ends of polyadenylated and capped transcripts (TSS-seq), approximately 7-9åÊë_g of poly(A)+ RNA together with inåÊvitro spike-ins was first subjected to zinc-mediated fragmentation (Ambion) at 70å¡C;Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems);Libraries were quantified by Qubit and sequenced on the HiSeq 4000 platform (Illumina), and typically generated ‰ö_39 million 76bp strand-specific single-end reads per sample;‰ö_16 million single-end reads were generated from the ‰ÛÏNo decapping‰Û control library.;developed the TSS-seq protocol and prepared the TSS-seq libraries;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "The radioactively labeled probes were synthesized using a Prime-it II Random Primer Labeling Kit (Agilent), 25åÊng of target-specific DNA template, and dATP [ë±-32P] (Perkin-Elmer or Hartmann Analytic);1åÊë_g of intact yeast total RNA was depleted of ribosomal RNA (rRNA) using a commercial kit (Illumina RiboZero Gold rRNA Removal Kit (Yeast)) for total RNA sequencing and 500åÊng of RNA was used for polyadenylated (polyA) RNA sequencing;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);First strand cDNA synthesis was performed using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMixPCR Kit (KAPA Biosystems) after RNase H (NEB) and RNase cocktail digestion (Ambion);Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems)" https://doi.org/10.1016/j.molcel.2018.10.018 TRUE
SAMN10435296 Illumina HiSeq 2500 sequencing; GSM3476453: spt6-1004 RNA Rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM3476453: spt6-1004 RNA Rep1; Saccharomyces cerevisiae; RNA-Seq 7/12/18 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR8198059 SAMN10435296 GSM3476453 spt6-1004 RNA Rep1 NA Saccharomyces cerevisiae PRJNA505786 GSE122620 Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction ftp.sra.ebi.ac.uk/vol1/fastq/SRR819/009/SRR8198059/SRR8198059_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR819/009/SRR8198059/SRR8198059_2.fastq.gz 12 TRUE "Dronamraju R et al., \Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction.\"", Cell Rep, 2018 Dec 18;25(12):3476-3489.e5""" Stranded "Stranded RNA sequencing (RNA-seq) analysis (with spike-in controls for normalization) was performed in WT, spt6S8‰ ÕE8, spt6S8‰ ÕA8, and spt6‰ÛÒ1004 strains;We further validated the occurrence of these antisense transcripts by strand-specific quantitative real-time PCR and determined whether they would be associated with nucleosome decreases at their 5‰Û_ ends;Stranded RNA-seq libraries were prepared using TruSeq Stranded Total RNA sample preparation according to manufacturer‰Ûªs instructions;Stranded RNA-seq allows us to map reads to specific strands, so all aligned reads were assigned sense or antisense based on whether they overlapped sacCer3 RefSeq genes in the same or opposite strand, respectively;Reads that didn‰Ûªt overlap any gene were discarded for any stranded analyses as we couldn‰Ûªt confidently assign them sense/antisense" "Here, we report that casein kinase II (CKII) phosphorylation of Spt6 is required for nucleosome occupancy at the 5‰Û_ ends of genes to prevent aberrant antisense transcription and enforce transcriptional directionality;Our results suggest that during transcription elongation, CKII phosphorylation of Spt6 facilitates the interaction with Spn1, which promotes nucleosome occupancy at the 5‰Û_ ends of genes to enforce the accuracy and directionality of transcription.;Mechanistically, we show that Spt6 phosphorylation is required for proper Spt6-Spn1 interaction, which we suggest plays a role in regulating the ability of Spt6 to deposit nucleosomes and enforce directionality of RNAPII, as represented in Figure 6." "ERCC spike-in controls were added to the RNA samples after rRNA clean-up and before proceeding on to the library preparation;Stranded RNA-seq libraries were prepared using TruSeq Stranded Total RNA sample preparation according to manufacturer‰Ûªs instructions;The libraries were sequenced on Illumina HiSeq 2500, paired-end 50bp reads);URL https://www.R-project.org/) was used for the 5‰Û_-half and 3‰Û_-half calculations, and R function ‰Û÷heatmap.2‰Û_ in the ‰Û÷gplots‰Ûª (https://cran.r-project.org/web/packages/gplots/index.html) library was used to plot the signal in the heatmap (in order of gene length);Libraries for the ChIP seq were prepared using Kappa hyper prep kit using manufacturer‰Ûªs instructions;To eliminate possible PCR artifacts from library preparation, we used in-house scripts to keep at most 5 reads that had the same sequence, where those above that threshold were filtered out;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Mutagenesis of pRS306-FLAG-SPT6, a gift from Fred Winston, Harvard Medical School, Boston, MA, (Kaplan et al., 2005) (Table S2) was performed with the QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) and primers described in Table S4;The isolated RNA was treated with 10 U of RNase-free DNase (Promega) for 30 minutes, followed by RNA cleanup (QIAGEN RNeasy Mini Kit, 74106);2.5 ë_g of total RNA was used to deplete rRNA using the Ribo-zero kit (Illumina);Low quality reads were then filtered with fastq_quality_filter, a function within the fastx-toolkit (v0.0.14), with command line options -p 90 and -q 20 to keep reads with at least a 20 Phred score at a minimum of 90% of the bases;Libraries for the ChIP seq were prepared using Kappa hyper prep kit using manufacturer‰Ûªs instructions;Low quality reads were then filtered with fastq_quality_filter, a function within the fastx-toolkit (v0.0.14), with command line options -p 90 and -q 20 to keep reads with at least a 20 Phred score at a minimum of 90% of the bases" https://doi.org/10.1016/j.celrep.2018.11.089 TRUE
SAMN03200216 Illumina HiSeq 2500 sequencing; GSM1550036: JKM139_1_T0; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM1550036: JKM139_1_T0; Saccharomyces cerevisiae; RNA-Seq 21/3/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR1656884 SAMN03200216 GSM1550036 JKM139_1_T0 NA Saccharomyces cerevisiae PRJNA267766 GSE63444 RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/004/SRR1656884/SRR1656884_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR165/004/SRR1656884/SRR1656884_2.fastq.gz 12 TRUE "Manfrini N et al., \Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae.\"", Elife, 2015 Jul 31;4Manfrini N et al., \""RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.\"", EMBO Rep, 2015 Feb;16(2):221-31""" Stranded (Truseq Stranded) "Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism;Since the DSB ends undergo nucleolytic degradation (resection) of their 5‰Û_-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition;Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S;Breaks that form across both strands in a DNA double helix are considered the most dangerous type of DNA damage, and can cause a cell to die or become cancerous if they are not repaired accurately.;‰Û÷Homologous recombination‰Ûª is one of the main mechanisms used by cells to repair DNA double-strand breaks;This mechanism requires enzymes to eat away at the end of one of the DNA strands on each side of the double-strand break;This process is called ‰Û÷resection‰Ûª and it exposes single strands of DNA;These single-stranded DNA ‰Û÷tails‰Ûª are then free to interact with an intact copy of the same DNA sequence from elsewhere in the cell's nucleus, which is used as a guide when repairing the damage.;Previous research has reported that forming a double-strand break in the DNA reduces the levels of transcription for the genes that surround the break, but it was not clear how this occurred.;In mammalian cells, inhibiting the transcription of genes around a double-strand DNA break depends on a signaling pathway that is activated whenever DNA damage is detected;Instead, the experiments indicate that it is the resection of the DNA around a double-strand break to form single-stranded tails that inhibits transcription in budding yeast;One of the next challenges will be to see if the resection process makes any contribution to changes in the transcription of genes that surround a double-strand break in mammals as well.;DNA double-strand breaks (DSBs) are particularly dangerous for cells, since their inefficient or inaccurate repair can result in deletions and chromosomal translocations that can lead to cancer and/or severe developmental abnormalities in humans;One of the main mechanisms to repair DSBs is homologous recombination (HR), which requires resection of the broken ends in order to generate 3‰Û_-ended single-stranded DNA (ssDNA) tails that invade the homologous undamaged template;The 5‰Û_-ending strands can then be further degraded by two other machineries depending on Exo1 and Sgs1-Dna2, respectively (Symington and Gautier, 2011).;cerevisiae cells is due to the conversion of DSB ends from double-stranded DNA (dsDNA) to ssDNA.;(A) Strand-specific RNA-seq data from the two biological replicates of wild type strain (JKM139) before (time zero, T0) and 60 (T60) or 240 (T240) min after HO induction, were uniquely mapped to the MAT locus å±10 kb;For each time point, densities (tag/nucleotide, log2 scale) from the two replicates were pooled and visualized as a heatmap with the upper and lower panels corresponding to the + and ‰öÕ strands, respectively;The level of these mRNAs decreased progressively as the distance of the corresponding genes from the DSB diminished and such decrease was independent of the strand that was transcribed (Figure 2A);Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded MAT probe (ss probe) that anneals to the unresected strand;Genomic DNA prepared from samples collected in (A) was analysed for single-stranded DNA (ssDNA) formation at the indicated times after HO induction as described in (B);Resection of the DSB 5‰Û_ strand can be measured by following the loss of restriction fragments by Southern blot analysis with a ssRNA probe annealing on one side of the break (Figure 5B);Concomitantly with the progression of 5‰Û_‰ÛÒ3‰Û_ resection (Figure 6A), also the binding of Rpb2 to distal genes progressively diminished (Figure 6B), suggesting that the decrease of RNA polymerase II occupancy around the DSB correlates with the time it takes for a DNA-end to become single-stranded.;cerevisiae is initiated by MRX and Sae2, which catalyze an endonucleolytic cleavage of the 5‰Û_ strands;The resulting partially resected 5‰Û_ strand can be further processed by the nucleases Exo1 and Dna2, the latter working in concert with the 3‰Û_‰ÛÒ5‰Û_ helicase Sgs1 (Mimitou and Symington, 2008; Zhu et al., 2008);Nucleolytic processing of the template DNA strand should stop transcription of the corresponding gene;However, we found that mRNA levels and RNA polymerase II occupancy around a DSB decrease independently of the strand that is transcribed, indicating that loss of non-template DNA strands also impedes transcription;Consistent with this hypothesis, it has been shown that within a region containing a stretch of dsDNA preceding a single-strand 3‰Û_ DNA end, purified RNA polymerase II transcribes within the dsDNA portion (Lilley and Houghton, 1979; Kadesch and Chamberlin, 1982);Furthermore, structural analyses of transcription initiation reveals that RNA polymerase II and its associated General Transcription Factors bind double-stranded promoter DNA to form a closed preinitiation complex (PIC), where the transcriptional machinery interacts with both template and non-template DNA strands (Liu et al., 2013; Sainsbury et al., 2015);Moreover, removal of either the template or non-template strands prevents binding of T7 RNA polymerase to the promoter, supporting the importance of protein-dsDNA interaction in the spatial organization of the transcriptional initiation complex (Maslak and Martin, 1993).;In fact, the double-stranded promoter DNA follows a straight path in the PIC complex and this rigidity allows the subsequent transition from a closed to an open promoter complex, where a central DNA region is melted leading to a transcription bubble in which the DNA template strand enters the RNA polymerase II cleft (Murakami et al., 2013);Furthermore, competition between the non-template DNA strand and the RNA transcript for base-pairing with the DNA template strand was shown to be important for maintaining structure and function during elongation (Kireeva et al., 2011), reinforcing the inhibitory effects of ssDNA on the transcription process.;Resection of the 5‰Û_ strands at both DSB ends leads to release of the transcription machinery (dashed lines) and to subsequent transcription arrest independently of whether the degraded DNA strand acts as template or non-template;Since the RNA polymerase binds double-stranded promoter DNA, generation of ssDNA at the DSB ends prevents reinitiation events (bar-headed line);DSB end resection at the MAT locus in JKM139 derivative strains was analyzed on alkaline agarose gels as previously described (Clerici et al., 2008), by using a single-stranded probe complementary to the unresected DSB strand;First strand cDNA synthesis was performed with the Bio-Rad iScript cDNA Synthesis Kit;Thank you for submitting your work entitled ‰ÛÏResection drives transcriptional repression around a DNA double-strand break in Saccharomyces cerevisiae‰Û for peer review at eLife;3) The result with the mec1‰ö tel1‰ö double mutant needs to be reported in order to conclude that the DNA damage checkpoint is not required for stopping transcription around a DNA double strand break.;Although brief, this is an important study that makes significant advances on the previous work from Jim Haber's lab showing that there is progressive transcriptional repression around the DNA double strand break (DSB) induced by the HO endonuclease at the yeast MAT locus;2) Mechanically, it makes sense that DNA resection would block transcription, given that RNA polymerase II binds to double strand DNA, and cannot transcribe from single strand DNA;This is a very straightforward analysis of a consequence of DNA double-strand break on local transcription;At the risk of oversimplification, the message from this story seems to be very simply simplified, that conversion of DNA from double strand to single strand, that allows homologous recombination, has an ‰Û÷unintended‰Ûª consequence; genes that become single-stranded can no longer by transcribed (presumably it‰Ûªs well know that RNA polymerase binding and regulation etc;occurs only on double-strand templates, and not on single-strand templates);Yet, most critically is the double-strand to single-strand conversion as the mechanistic explanation;3) They need a citation that in fact single-strand templates do not support transcription.;Resection of double-stranded DNA will prevent transcription factors (such as TBP) from binding to promoter elements and as a result, transcription is no longer possible (and of course, depending on the orientation of the gene, resection can mean that there is no longer a template for the RNA polymerase!);shown in Figure 1), the only genes affected are precisely those genes that would no longer be completely double stranded in many cells;that loss of the double stranded DNA sequence results in loss of binding by RNA polymerase II).;3) The result with the mec1‰ö tel1‰ö double mutant needs to be reported in order to conclude that the DNA damage checkpoint is not required for stopping transcription around a DNA double strand break.;Although brief, this is an important study that makes significant advances on the previous work from Jim Haber's lab showing that there is progressive transcriptional repression around the DNA double strand break (DSB) induced by the HO endonuclease at the yeast MAT locus;Mechanically, it makes sense that DNA resection would block transcription, given that RNA polymerase II binds to double strand DNA, and cannot transcribe from single strand DNA;Yet, most critically is the double-strand to single-strand conversion as the mechanistic explanation;3) They need a citation that in fact single-strand templates do not support transcription.;Resection of double-stranded DNA will prevent transcription factors (such as TBP) from binding to promoter elements and as a result, transcription is no longer possible (and of course, depending on the orientation of the gene, resection can mean that there is no longer a template for the RNA polymerase!);shown in Figure 1), the only genes affected are precisely those genes that would no longer be completely double stranded in many cells;that loss of the double stranded DNA sequence results in loss of binding by RNA polymerase II)." Blue arrows indicate direction of transcription. "Total RNA-seq libraries were previously described (Manfrini et al., 2015) and data were retrieved from the Gene Expression Omnibus (accession number {\type\"":\""entrez-geo\"",\""attrs\"":{\""text\"":\""GSE63444\"",\""term_id\"":\""63444\""}}GSE63444; Manfrini et al., 2014);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA """ "Total RNA was extracted from cells using the Bio-Rad (Hercules, CA) Aurum total RNA mini kit;First strand cDNA synthesis was performed with the Bio-Rad iScript cDNA Synthesis Kit" https://doi.org/10.7554/eLife.08942 TRUE
SAMN04505769 Illumina HiSeq 2500 sequencing; GSM2067892: wild type rep 1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2067892: wild type rep 1; Saccharomyces cerevisiae; RNA-Seq 1/3/16 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR3180780 SAMN04505769 GSM2067892 wild type rep 1 NA Saccharomyces cerevisiae PRJNA312727 GSE78136 Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast ftp.sra.ebi.ac.uk/vol1/fastq/SRR318/000/SRR3180780/SRR3180780_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR318/000/SRR3180780/SRR3180780_2.fastq.gz 18 TRUE "Masser AE et al., \Cytoplasmic protein misfolding titrates Hsp70 to activate nuclear Hsf1.\"", Elife, 2019 Sep 25;8Gowda NK et al., \""Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast.\"", Mol Biol Cell, 2016 Apr 15;27(8):1210-9""" Stranded (Truseq Stranded) Libraries were prepared using Illumina TruSeq Stranded mRNA (polyA selection) and quality checked using Quant-iT (DNA BR) and CaliperGX "Libraries were prepared using Illumina TruSeq Stranded mRNA (polyA selection) and quality checked using Quant-iT (DNA BR) and CaliperGX;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Samples were separated by SDS-PAGE and visualized by Pierce(TM) Silver Staining Kit (Thermo Fisher).;RNA was extracted from cells grown in YPD using a RiboPure RNA Purification Kit for Yeast (Ambion, Invitrogen);cDNA was synthesized from DNase I-treated RNA using Superscript III Reverse Transcriptase (Invitrogen) and qPCR was performed using KAPA SYBR Fast Universal qPCR Kit (KAPA Biosystems) with primers listed Table 3;Cells were harvested by centrifugation and RNA was extracted using the RiboPure RNA Purification Kit for Yeast (Thermo Fisher Scientific);0.5% (5 ë_l) and 1.25% (1 ë_l) of the respective soluble and pelleted fractions were analyzed by SDS-PAGE and proteins were stained using the by Pierce Silver Staining Kit (Thermo Fisher)." https://doi.org/10.7554/eLife.47791 TRUE
SAMN11191529 "Illumina HiSeq 4000 sequencing; GSM3682096: Naive Diploid mRNA-seq, replicate 1; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 4000 sequencing; GSM3682096: Naive Diploid mRNA-seq, replicate 1; Saccharomyces cerevisiae; RNA-Seq" 1/3/20 Illumina HiSeq 4000 ILLUMINA PAIRED cDNA SRR8761856 SAMN11191529 GSM3682096 "Naive Diploid mRNA-seq, replicate 1" NA Saccharomyces cerevisiae PRJNA528474 GSE128672 Transcription Profiles of [ESI+] and Set3C loss of function ftp.sra.ebi.ac.uk/vol1/fastq/SRR876/006/SRR8761856/SRR8761856_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR876/006/SRR8761856/SRR8761856_2.fastq.gz 20 TRUE "Harvey ZH et al., \A Prion Epigenetic Switch Establishes an Active Chromatin State.\"", Cell, 2020 Mar 5;180(5):928-940.e14""" Unstranded (e NEBNext Ultra II DNA Library Prep Kit) NA NA NA NA FALSE FALSE
SAMN08595322 Illumina HiSeq 2500 sequencing; GSM3021114: WT_10H2 log; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM3021114: WT_10H2 log; Saccharomyces cerevisiae; RNA-Seq 7/12/18 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR6767450 SAMN08595322 GSM3021114 WT_10H2 log NA Saccharomyces cerevisiae PRJNA435723 GSE111056 Impact of intron deletion on starvation dependent repression of gene expression ftp.sra.ebi.ac.uk/vol1/fastq/SRR676/000/SRR6767450/SRR6767450_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR676/000/SRR6767450/SRR6767450_2.fastq.gz 12 TRUE "Parenteau J et al., \Introns are mediators of cell response to starvation.\"", Nature, 2019 Jan;565(7741):612-617""" Stranded (Illumina ScriptSeq RNA-seq library preparation kit ) NA NA NA NA FALSE FALSE
SAMN12107237 Illumina HiSeq 2500 sequencing; GSM3900812: S3_SS: ind ret 1-1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM3900812: S3_SS: ind ret 1-1; Saccharomyces cerevisiae; RNA-Seq 28/2/20 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR9336459 SAMN12107237 GSM3900812 S3_SS: ind ret 1-1 NA Saccharomyces cerevisiae PRJNA550078 GSE133136 "Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: slow growth, low pH and high CO2 levels" ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/009/SRR9336459/SRR9336459_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/009/SRR9336459/SRR9336459_2.fastq.gz 18 TRUE "Hakkaart X et al., \Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels.\"", Biotechnol Bioeng, 2020 Mar;117(3):721-735""" Stranded ( using a 250_300 bp insert strand specific library which was prepared by Novogene) "Notable differences between laboratory and industrial conditions included the regulation of PDR12, which encodes a plasma‰Ûmembrane transporter in weak organic acid tolerance (Piper et al., 1998; Ullah, Orij, Brul, & Smits, 2012), that responded in opposite directions under the two conditions, and the enrichment of genes encoding extracellular proteins and/or involved in cell wall processes among the genes whose expression was positively correlated with increasing growth rate under laboratory conditions but not industrial conditions (Supporting Information Appendix 5)." "DE, differentially expressed; FC, fold change; FDR, false‰Ûdiscovery rate [Color figure can be viewed at wileyonlinelibrary.com];National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " https://doi.org/10.1002/bit.27210 TRUE
SAMN07709372 Illumina HiSeq 2000 sequencing; GSM2794870: H3D_H3H_0_rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM2794870: H3D_H3H_0_rep1; Saccharomyces cerevisiae; RNA-Seq 18/10/17 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR6080162 SAMN07709372 GSM2794870 H3D_H3H_0_rep1 NA Saccharomyces cerevisiae PRJNA412318 GSE104312 RNA transcription profile of different yeast mutants under glucose starvation (0.05% glucose) and comparison of transcriptome of WT and H3D_H3H ftp.sra.ebi.ac.uk/vol1/fastq/SRR608/002/SRR6080162/SRR6080162_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR608/002/SRR6080162/SRR6080162_2.fastq.gz 12 TRUE "Zhou Z et al., \Independent manipulation of histone H3 modifications in individual nucleosomes reveals the contributions of sister histones to transcription.\"", Elife, 2017 Oct 13;6""" Unclear (DNA library methods on RNA) "H3K4, H3K36 and H3K79 methylation affects DNA double-strand break (DSB) repair (Faucher and Wellinger, 2010; Jha and Strahl, 2014; Pai et al., 2014);The first and second strand cDNAs were synthesized usingåÊthe SuperScript III CellsDirect cDNA Synthesis Kit (Invitrogen) and theåÊSuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen), respectively;The resulting double-stranded DNA was subjected to DNA repair and end-polishing (blunt-end) usingåÊthe End-It DNA End-Repair Kit (Epicentre)" Bidirectional arrows with gradient colors indicate the increasing skewness of log2 fold change in asymmetrical K4R mutants to those in either H3D/H3H (red end) or H3DK4R/H3HK4R (blue end) cells;(B) Bidirectional arrows with gradient colors indicate the increasing skewness of log2 fold-change in asymmetrical K4R mutants to those in either H3D/H3H (red end) or H3DK4R/H3HK4R (blue end) cells "The method for constructing RNA-Seq libraries was modified from the TruSeq DNA sample preparation kit protocol (Illumina);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Total RNA was isolated from yeast cells with an RNeasy mini kit (Qiagen);cDNA was synthesized using theåÊFastquant RT kit (Tiangen);Total RNA was extracted usingåÊthe Yeast RNA extraction kit (Qiagen), resolved on agarose-formaldehyde gels and transferred to Hybond-N+ membrane (GE);The method for constructing RNA-Seq libraries was modified from the TruSeq DNA sample preparation kit protocol (Illumina);Briefly, total RNA was isolated usingåÊthe RNeasy midi kit (Qiagen);The first and second strand cDNAs were synthesized usingåÊthe SuperScript III CellsDirect cDNA Synthesis Kit (Invitrogen) and theåÊSuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen), respectively;The resulting double-stranded DNA was subjected to DNA repair and end-polishing (blunt-end) usingåÊthe End-It DNA End-Repair Kit (Epicentre);The DNA was then purified with theåÊQIAquick PCR Purification Kit (Qiagen) and a dA-tail was added using the 3'‰öÕ5' exo-Klenow Fragment (NEB);The 200‰ÛÒ500 bp ligation products were recovered from a 2% (w/v) agarose gel using theåÊQiagen gel extraction kit and were PCR amplified with Illumina primers using theåÊKAPA HiFiåÊHotStart kit;We used theåÊFASTX Toolkit (RRID:SCR_005534) to remove the adapter sequences" https://doi.org/10.7554/eLife.30178 TRUE
SAMN14007540 Illumina HiSeq 3000 sequencing; GSM4297054: glu_6h; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 3000 sequencing; GSM4297054: glu_6h; Saccharomyces cerevisiae; RNA-Seq 21/2/20 Illumina HiSeq 3000 ILLUMINA PAIRED cDNA SRR11029235 SAMN14007540 GSM4297054 glu_6h NA Saccharomyces cerevisiae PRJNA605000 GSE144820 Adapting the 10x Genomics Platform for single-cell RNA-seq in yeast reveals the importance of stochastic gene expression during the lag phase ftp.sra.ebi.ac.uk/vol1/fastq/SRR110/035/SRR11029235/SRR11029235.fastq.gz 30 TRUE "Jariani A et al., \A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast.\"", Elife, 2020 May 18;9""" UNCLEAR "A more recent study that introduced a clever method for strand-specific detection of transcripts in single yeast cells studied 285 single Saccharomyces cerevisiae yeast cells grown in rich media, and detected on average 3339 transcripts (Nadal-Ribelles et al., 2019)." "As detailed in the methods section, all samples were processed on the 10x Genomics platform and the generated cDNA libraries were sequenced using the Illumina platform;Moreover, the ability to increase sample size in droplet-based methods allows the library preparation cost to be greatly reduced to $0.3/cell compared to previously reported costs of $4.15/cell (Nadal-Ribelles et al., 2019);The prepared libraries were sequenced on one run of Illumina NextSeq and two lanes of Illumina HiSeq 3000;We thank the VIB Nucleomics Core (http://www.nucleomics.be/) for sequencing of the cDNA libraries;The technical advance of this manuscript is that this method would allow for a considerable scale-up (to 1500 cells) relative to previously 96 well plate methods and a reduction in cost for library preparation per cell (from $4.15 to $1.00, or even $0.30 if scaled up to 5,000 cells), which will open up new areas of research for yeast biologists;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Moreover, it should be noted that the newer chemistry (v3) of the 10x Genomics Chromium Single Cell 3‰Ûª Reagent kit has a higher mRNA yield compared to v2 chemistry which is used in our study;We used the Chromium Single Cell 3‰Û_ kit (v2) of 10x Genomics for scRNA-seq;This volume and the cell counts were chosen based on a target of 1000 cells for the mix-glucose-maltose condition and 2000 cells for the rest of the samples, according to the manual of single-cell 3‰Ûª kit" https://doi.org/10.7554/eLife.55320 TRUE
SAMN10290909 Illumina HiSeq 2500 sequencing; GSM3445806: YSB787-Ra-0; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM3445806: YSB787-Ra-0; Saccharomyces cerevisiae; RNA-Seq 23/7/19 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR8109496 SAMN10290909 GSM3445806 YSB787-Ra-0 NA Saccharomyces cerevisiae PRJNA498394 GSE121762 Transcription-dependent targeting of Hda1C to hyperactive genes mediates H4-specific deacetylation in yeast (RNA-seq) ftp.sra.ebi.ac.uk/vol1/fastq/SRR810/006/SRR8109496/SRR8109496_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR810/006/SRR8109496/SRR8109496_2.fastq.gz 16 TRUE "Ha SD et al., \Transcription-dependent targeting of Hda1C to hyperactive genes mediates H4-specific deacetylation in yeast.\"", Nat Commun, 2019 Sep 19;10(1):4270""" Stranded "Furthermore, the DNA-binding domains of Hda2 and Hda3 are able to bind to double- or single-stranded DNA in vitro11; however, their functions in vivo remain unknown.;A previous study showed that Hda2 and Hda3 can bind to double- and single-stranded DNA in vitro11;RNA samples from the time-course experiments described in a were analyzed by strand-specific RNA-sequencing;To explore how Hda1C affects global gene expression dynamics, total RNAs from the wild-type and hda1‰ö cells were analyzed by strand-specific RNA-sequencing;Total RNAs was treated with DNase I (Thermo Fisher Scientific) and first-strand cDNA was prepared with 1‰Ûäë_g total RNA, ReverTra Ace qPCR RT kit (TOYOBO), and gene-specific primers;Strand-specific probes were generated by unidirectional PCR in the presence of [ë±-32P] dATP with only one primer" Strand-specific probes were generated by unidirectional PCR in the presence of [ë±-32P] dATP with only one primer;The mRNA-seq libraries were prepared using NEXTflex Rapid Directional mRNA-Seq Kit (Bioo Scientific) and sequenced on the HiSeq2500 sequencer (Illumina) "The DNA libraries for ChIP-seq were prepared using Accel-NGS 2‰ÛäS Plus DNA Library Kit (Swift Biosciences) and sequenced on the HiSeq2500 platform (Illumina) following the manufacturer‰Ûªs instructions.;The mRNA-seq libraries were prepared using NEXTflex Rapid Directional mRNA-Seq Kit (Bioo Scientific) and sequenced on the HiSeq2500 sequencer (Illumina);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Total RNAs was treated with DNase I (Thermo Fisher Scientific) and first-strand cDNA was prepared with 1‰Ûäë_g total RNA, ReverTra Ace qPCR RT kit (TOYOBO), and gene-specific primers;The DNA libraries for ChIP-seq were prepared using Accel-NGS 2‰ÛäS Plus DNA Library Kit (Swift Biosciences) and sequenced on the HiSeq2500 platform (Illumina) following the manufacturer‰Ûªs instructions.;The mRNA-seq libraries were prepared using NEXTflex Rapid Directional mRNA-Seq Kit (Bioo Scientific) and sequenced on the HiSeq2500 sequencer (Illumina)" http://dx.doi.org/10.1038/s41467-019-12077-w TRUE
SAMN12349602 Illumina HiSeq 3000 sequencing; GSM3968534: WT_total mRNA_rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 3000 sequencing; GSM3968534: WT_total mRNA_rep1; Saccharomyces cerevisiae; RNA-Seq 6/1/20 Illumina HiSeq 3000 ILLUMINA PAIRED cDNA SRR9822680 SAMN12349602 GSM3968534 WT_total mRNA_rep1 NA Saccharomyces cerevisiae PRJNA556393 GSE134774 mSeq of total and polysome bound mRNAs in two yeast strains ftp.sra.ebi.ac.uk/vol1/fastq/SRR982/000/SRR9822680/SRR9822680_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR982/000/SRR9822680/SRR9822680_2.fastq.gz 12 TRUE "Shankar V et al., \rRNA expansion segment 27Lb modulates the factor recruitment capacity of the yeast ribosome and shapes the proteome.\"", Nucleic Acids Res, 2020 Apr 6;48(6):3244-3256""" NA "One microgram of total RNA and 1 ë_g polysome-associated RNA, isolated from polysome profile fractions, were poly(A) enriched and a cDNA library was prepared and sequenced using standard protocols for Illumina HiSeq3000;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkaa003 TRUE
SAMN06819213 Illumina HiSeq 2000 sequencing; GSM2586852: YO1829_YSEQ164; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM2586852: YO1829_YSEQ164; Saccharomyces cerevisiae; RNA-Seq 27/4/17 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR5468230 SAMN06819213 GSM2586852 YO1829_YSEQ164 NA Saccharomyces cerevisiae PRJNA383899 GSE98079 Transcriptional Profiling of Biofilm Regulators Identified by an Overexpression Screen in S. cerevisiae ftp.sra.ebi.ac.uk/vol1/fastq/SRR546/000/SRR5468230/SRR5468230_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR546/000/SRR5468230/SRR5468230_2.fastq.gz 20 TRUE "Cromie GA et al., \Transcriptional Profiling of Biofilm Regulators Identified by an Overexpression Screen in Saccharomyces cerevisiae.\"", G3 (Bethesda), 2017 Aug 7;7(8):2845-2854""" Stranded "Five micrograms of total RNA for each sample was then processed using the Tru-Seq stranded mRNA kit (Illumina) following manufacturer instructions;Briefly, we isolated total RNA from single colonies growing on solid medium and prepared libraries to measure stranded mRNA using the Illumina TruSeq method (Materials and Methods)" "Similarly, although there was a very poor correlation between the common factor and the TOS8 profile across all genes (R2 = 0.04) (Figure 4B), genes that were significantly (P < 0.01, after multiple hypothesis correction) differentially expressed in both profiles tended to show a consistent direction of effect (Fisher‰Ûªs exact test, 2-tailed: P < 2.2e‰öÕ16);However, among the overlap between these two profiles, there is a statistically significant overrepresentation of genes showing differential expression with the same direction of effect, i.e., induced or repressed in both profiles, compared to genes that have opposite directions of effect" "In order to perform the overexpression screen using the MoBY plasmid library (Ho et al;2009), we first created pools of plasmids by pinning the master library from 96-well plates to selective (G418) LB Lennox Omnitrays and scraping the colonies from 10 such pinned plates into each pool pellet;Individual sequencing libraries were pooled and analyzed by paired-end, 51 nucleotide read sequencing in one lane of an Illumina HiSeq 2000.;Library sizes were normalized using calcNormFactors, and dispersion parameters were estimated using the estimateGLMTrendedDisp and estimateGLMTagwiseDisp commands;2013) with the MoBY plasmid collection, a low copy number (CEN) plasmid library containing 4981 individual ORFs under the transcriptional control of their native promoters (Ho et al;Only 18 of these plasmids were identified more than once, suggesting either a high frequency of false positives or incomplete library coverage;Briefly, we isolated total RNA from single colonies growing on solid medium and prepared libraries to measure stranded mRNA using the Illumina TruSeq method (Materials and Methods);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " Five micrograms of total RNA for each sample was then processed using the Tru-Seq stranded mRNA kit (Illumina) following manufacturer instructions https://doi.org/10.1534/g3.117.042440 TRUE
SAMN03076101 "Illumina HiSeq 2500 sequencing; GSM1510523: phm4_del, high Pi, 0 min; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2500 sequencing; GSM1510523: phm4_del, high Pi, 0 min; Saccharomyces cerevisiae; RNA-Seq" 21/3/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR1583841 SAMN03076101 GSM1510523 "phm4_del, high Pi, 0 min" NA Saccharomyces cerevisiae PRJNA261797 GSE61667 Transcription profile of ?phm4 strain during growth in no phosphate medium ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/001/SRR1583841/SRR1583841_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/001/SRR1583841/SRR1583841_2.fastq.gz 19 TRUE "Vardi N et al., \Sequential feedback induction stabilizes the phosphate starvation response in budding yeast.\"", Cell Rep, 2014 Nov 6;9(3):1122-34""" NA NA NA NA NA https://doi.org/10.1016/j.celrep.2014.10.002 FALSE
SAMN08451834 "Illumina HiSeq 2500 sequencing; GSM2976206: FW5138_Rap1-AID degron induction 2 hours rescue Rap1 (1-600, CTD_del) total RNA-seq biological replicate 1; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2500 sequencing; GSM2976206: FW5138_Rap1-AID degron induction 2 hours rescue Rap1 (1-600, CTD_del) total RNA-seq biological replicate 1; Saccharomyces cerevisiae; RNA-Seq" 22/12/18 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR6662199 SAMN08451834 GSM2976206 "FW5138_Rap1-AID degron induction 2 hours rescue Rap1 (1-600, CTD_del) total RNA-seq biological replicate 1" NA Saccharomyces cerevisiae PRJNA432633 GSE110003 Identification of non-coding transcripts regulated by Rap1 and other transcription factors by RNA-seq analysis ftp.sra.ebi.ac.uk/vol1/fastq/SRR666/009/SRR6662199/SRR6662199_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR666/009/SRR6662199/SRR6662199_2.fastq.gz 24 TRUE "Wu ACK et al., \Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor.\"", Mol Cell, 2018 Dec 20;72(6):942-954.e7""" Stranded "Normalized reads (y axis) for the Watson (W, blue) and Crick (C, red) strands.;nåÊ= 564 Rap1 binding sites, signals for W and C strands were computed separately.;(E) Similar to (D), using scatterplots to display expression changes for antisense (AS) and sense (S) strand windows relative to the coding gene for Rap1 (nåÊ= 141) and Ume6 (nåÊ= 87) -regulated promoters;(F) Heatmaps showing changes in RNA expression on AS or S strands for data described in (E);TSS-seq signal per million reads and total RNA-seq data are plotted for both strands.;Changes in RNA expression were calculated for nåÊ= 141 promoter Rap1 sitesåÊå±100åÊbp for AS and S strands;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);Each library was sequenced on the HiSeq 2500 or 4000 platform (Illumina) and generated ‰ö_45 million 101åÊbp strand-specific paired-end reads per sample, on average.;First strand cDNA synthesis was performed using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMixPCR Kit (KAPA Biosystems) after RNase H (NEB) and RNase cocktail digestion (Ambion);Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems);Libraries were quantified by Qubit and sequenced on the HiSeq 4000 platform (Illumina), and typically generated ‰ö_39 million 76bp strand-specific single-end reads per sample;Fragment counts within specified windows (e.g.,åÊå± 100åÊbp) around the 564 Rap1 sites (1128 intervals total, Watson and Crick strand alignments were assigned to separate intervals) were obtained using the featureCounts tool from the Subread package (version 1.5.1) (Liao etåÊal., 2014);The parameters used were ‰ÛÏ-O‰ÛÒminOverlap 1‰ÛÒnonSplitOnly‰ÛÒprimary -s 2 -p -B -P -d 0 -D 600 -C.‰Û Windows on separate strands were treated as separate intervals for all strand-specific RNA-seq experiments, and only reads which overlapped with the corresponding strand and interval were counted;Uniquely mapped alignments corresponding to the sense and antisense strands were obtained using SAMtools view (version 1.3.1) by using the flags ‰ÛÏ-q 1 -F 20‰Û and ‰ÛÏ-q 1 -f 16,‰Û respectively (Li etåÊal., 2009);To calculate TPM values for each TSS, TSS-seq counts were obtained by quantifying the abundance of reads with the 1st transcribed 5‰Û_ nucleotide withinåÊå± 75åÊbp of annotated TSSs (Park etåÊal., 2014), on the respective strand" "Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction;We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.;The transcriptionally active coding gene promoters, which often express noncoding transcripts in the antisense direction (Neil etåÊal., 2009, Seila etåÊal., 2008, Xu etåÊal., 2009), are a major source;This process is known as divergent or bidirectional transcription;A large fraction of all noncoding transcripts emanate from divergent or bidirectional gene promoters (Neil etåÊal., 2009, Seila etåÊal., 2008, Xu etåÊal., 2009);Typically, divergent noncoding transcripts initiate within or nearby coding gene promoters, but they do not share the same core promoter as transcripts in the coding direction (Andersson etåÊal., 2015, Duttke etåÊal., 2015, Rhee and Pugh, 2012, Scruggs etåÊal., 2015);We find that depletion of Rap1, but not other transcription factors important for RP expression, causes transcription in the divergent direction;Rap1 represses noncoding transcription typically within 50åÊbp of the Rap1 motif, which is uncoupled from transcription regulation in the protein-coding direction;Thus, a sequence-specific transcription factor controls promoter directionality by repressing transcription in the divergent direction;Our work adds a new layer of regulation to various mechanisms that limit expression of aberrant transcripts and defines how promoter directionality is controlled.;In budding yeast, a large fraction of bidirectional promoters express noncoding transcripts, also known as cryptic unstable transcripts (CUTs) or stable unannotated transcripts (SUTs), in the divergent direction (Neil etåÊal., 2009, Xu etåÊal., 2009);Transcription of divergent CUTs and SUTs typically correlates with nucleosome-depleted regions (NDRs) and promoter activity in the coding gene direction;Expression near the Rap1 binding sites was upregulated in both the sense and antisense direction after Rap1 depletion; however, the largest increase was detected in the antisense direction (FigureåÊ2E);When we clustered for the sense direction signals, we observed that transcripts were upregulated both up- and downstream of the Rap1 site (Sc1 and Sc2);Thus, Rap1 represses transcription near the Rap1 binding sites in the antisense, and to lesser extent, the sense direction.;We integrated Rap1 motifs in a fluorescent reporter construct that harbors a divergent promoter transcribing PPT1 in the coding direction and SUT129 in the noncoding direction (pPS) (Figures 3D and S3A) (Marquardt etåÊal., 2014);The repression of SUT129 by Rap1 was not dependent on transcription regulation in the coding direction because in RAP1-AID (IAA or NT) cells the PPT1 signal matched WT (FigureåÊ3E, right panel);As expected, TSS signals decreased in the sense direction downstream of the Rap1 binding sites in Rap1-depleted cells because coding gene expression was reduced (FigureåÊ4B, sense);Interestingly, sequences directly upstream of the canonical coding transcript TSSs displayed increased TSS signals in the sense direction, suggesting that Rap1 is also important for TSS selection (Challal etåÊal., 2018);Rap1 promotes transcription from the coding direction TSS (black arrows) but represses initiation from the divergent TSS;When Rap1 is absent, transcription in the divergent direction occurs.;Our data demonstrate how a sequence-specific transcription factor can prevent regulatory sequences from producing aberrant transcripts and define a mechanism for providing directionality toward productive transcription.;Several lines of evidence indicate that Rap1 represses divergent noncoding transcription, uncoupled from transcription regulation in the coding direction;Fourth, the Rap1 binding site ectopically represses divergent noncoding transcription without affecting transcription in the protein-coding direction;Conversely, the AD of Rap1, which directs transcription in the protein-coding direction, is not required for repressing divergent transcription;Promoter directionality is shaped by evolution toward protein-coding genes through enrichment of DNA binding protein motifs (Jin etåÊal., 2017);In this context, Rap1 promotes directionality in multiple ways;First, Rap1 recruits cofactors and basal transcription machinery, which promote transcription in the coding direction (Azad and Tomar, 2016, Hu and Li, 2007);Core promoters are intrinsically directional (Duttke etåÊal., 2015), and two independent pre-initiation complexes initiate divergent transcription at mRNA-noncoding RNA pairs in yeast (Rhee and Pugh, 2012);Hence, repressing transcription initiation at the antisense core promoter regulates promoter directionality;Our data indicate that Rap1 restricts RSC to stimulate productive transcription in the protein-coding direction" "Total RNA from yeast was incubated with rDNase (Machery-Nagel) and column purified (Machery-Nagel) prior to sequencing library preparation;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);Each library was sequenced on the HiSeq 2500 or 4000 platform (Illumina) and generated ‰ö_45 million 101åÊbp strand-specific paired-end reads per sample, on average.;To obtain libraries representing the 5‰Û_ ends of polyadenylated and capped transcripts (TSS-seq), approximately 7-9åÊë_g of poly(A)+ RNA together with inåÊvitro spike-ins was first subjected to zinc-mediated fragmentation (Ambion) at 70å¡C;Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems);Libraries were quantified by Qubit and sequenced on the HiSeq 4000 platform (Illumina), and typically generated ‰ö_39 million 76bp strand-specific single-end reads per sample;‰ö_16 million single-end reads were generated from the ‰ÛÏNo decapping‰Û control library.;developed the TSS-seq protocol and prepared the TSS-seq libraries;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "The radioactively labeled probes were synthesized using a Prime-it II Random Primer Labeling Kit (Agilent), 25åÊng of target-specific DNA template, and dATP [ë±-32P] (Perkin-Elmer or Hartmann Analytic);1åÊë_g of intact yeast total RNA was depleted of ribosomal RNA (rRNA) using a commercial kit (Illumina RiboZero Gold rRNA Removal Kit (Yeast)) for total RNA sequencing and 500åÊng of RNA was used for polyadenylated (polyA) RNA sequencing;Libraries were prepared using the TruSeq Stranded Total RNA kit or TruSeq stranded mRNA kit (Illumina) according to the manufacturer‰Ûªs instructions (10 or 13 PCR cycles);First strand cDNA synthesis was performed using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific), and second strand synthesis was performed using a KAPA HiFi HotStart ReadyMixPCR Kit (KAPA Biosystems) after RNase H (NEB) and RNase cocktail digestion (Ambion);Double-stranded cDNA was quantified by Qubit fluorometric quantitation (Thermo Fisher Scientific) and used as inputs for library preparation using a KAPA Hyper Prep Kit (KAPA Biosystems) and KAPA Single-Indexed adapters for Illumina platforms (KAPA Biosystems)" https://doi.org/10.1016/j.molcel.2018.10.018 TRUE
SAMN12534257 "Illumina HiSeq 2000 sequencing; GSM4017981: BY4741 wt yeast cells, 30å¼C , RNAseq 1; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2000 sequencing; GSM4017981: BY4741 wt yeast cells, 30å¼C , RNAseq 1; Saccharomyces cerevisiae; RNA-Seq" 23/11/19 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR9929263 SAMN12534257 GSM4017981 "BY4741 wt yeast cells, 30å¼C , RNAseq 1" NA Saccharomyces cerevisiae PRJNA559331 GSE135568 A multi-omics dataset of heat-shock response in the yeast RNA transport protein Mip6 ftp.sra.ebi.ac.uk/vol1/fastq/SRR992/003/SRR9929263/SRR9929263_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR992/003/SRR9929263/SRR9929263_2.fastq.gz 24 TRUE "Nu̱o-Cabanes C et al., \A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6.\"", Sci Data, 2020 Feb 27;7(1):69""" NA "However, the direction of signal change at the gene-level seems to be the same for both omics layers, since the position of selected genes in the Log2FC distributions is similar for RNA-seq and H4K12ac data (Fig.åÊ7c)" "National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " http://dx.doi.org/10.1038/s41597-020-0412-z TRUE
SAMN05559818 Illumina HiSeq 2500 sequencing; GSM2267326: WT Snf2 RNAseq Replicate 1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2267326: WT Snf2 RNAseq Replicate 1; Saccharomyces cerevisiae; RNA-Seq 1/12/16 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR4018567 SAMN05559818 GSM2267326 WT Snf2 RNAseq Replicate 1 NA Saccharomyces cerevisiae PRJNA338508 GSE85460 Loss of Snf5 and the formation of an aberrant SWI/SNF complex ftp.sra.ebi.ac.uk/vol1/fastq/SRR401/007/SRR4018567/SRR4018567_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR401/007/SRR4018567/SRR4018567_2.fastq.gz 12 TRUE "Sen P et al., \Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.\"", Cell Rep, 2017 Feb 28;18(9):2135-2147""" Stranded (Truseq Stranded mRNA LT Sample Preparation kit) NA NA NA NA http://twitter.com/intent/tweet?text=Aurora%20A%20is%20a%20repressed%20effector%20target%20of%20the%20chromatin%20remodeling%20protein%20INI1/hSNF5%20required%20for%20rhabdoid%20tumor%20%E2%80%A6%20https%3A//pubmed.ncbi.nlm.nih.gov/21521802/ FALSE
SAMN09453376 Illumina HiSeq 4000 sequencing; GSM3204446: YMB10544_a; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 4000 sequencing; GSM3204446: YMB10544_a; Saccharomyces cerevisiae; RNA-Seq 26/6/18 Illumina HiSeq 4000 ILLUMINA PAIRED cDNA SRR7363059 SAMN09453376 GSM3204446 YMB10544_a NA Saccharomyces cerevisiae PRJNA476714 GSE115990 "SUMO-targeted ubiquitin ligases (STUbLs) reduce the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin" ftp.sra.ebi.ac.uk/vol1/fastq/SRR736/009/SRR7363059/SRR7363059_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR736/009/SRR7363059/SRR7363059_2.fastq.gz 11 TRUE "Ohkuni K et al., \SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin.\"", Front Genet, 2018;9:379""" Stranded Three independent RNA-seq libraries for each of 4 samples were prepared from total RNA using the Illumina TruSeq Stranded Total RNA Kit RS-122-2201 "Three independent RNA-seq libraries for each of 4 samples were prepared from total RNA using the Illumina TruSeq Stranded Total RNA Kit RS-122-2201;One library (YMB10544_c) contained 45% rRNA sequences and was removed from further analysis (all other libraries contained < 2% rRNA reads);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Yeast cells were transformed as previously described (Amberg et al., 2005) or using the frozen-EZ yeast transformation II kit (Zymo research corporation, Irvine CA);For mammalian 2-hybrid assays, the Matchmaker Mammalian Assay Kit 2 (Clontech.com Cat;The Great EscAPe Chemiluminescence kit (Clontech #631737) was used to detect SEAP levels in the mammalian 2-hybrid assay;Total RNAs were isolated from 3 OD600 equivalent cells using MasterPureTM Yeast RNA purification kit with DNase I treatment as indicated by the manufacturer (Epicentre);Three independent RNA-seq libraries for each of 4 samples were prepared from total RNA using the Illumina TruSeq Stranded Total RNA Kit RS-122-2201;Additionally, we stained cells with the LIVE/DEAD Yeast Viability Kit (Thermo Fisher) to quantitate dead or dying cells in the culture (dead)" https://doi.org/10.3389/fgene.2018.00379 TRUE
SAMN08567232 Illumina HiSeq 2000 sequencing; GSM3017184: GAL-REG (WT)-1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM3017184: GAL-REG (WT)-1; Saccharomyces cerevisiae; RNA-Seq 1/6/18 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR6749957 SAMN08567232 GSM3017184 GAL-REG (WT)-1 NA Saccharomyces cerevisiae PRJNA434650 GSE110818 RNA-seq of strains grown under the control of native galactose regulon or synthetic xylose regulon vs constitutive expression of galactose and xylose metabolic genes ftp.sra.ebi.ac.uk/vol1/fastq/SRR674/007/SRR6749957/SRR6749957_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR674/007/SRR6749957/SRR6749957_2.fastq.gz 12 TRUE "Endalur Gopinarayanan V et al., \A semi-synthetic regulon enables rapid growth of yeast on xylose.\"", Nat Commun, 2018 Mar 26;9(1):1233""" NA "Next, we developed a selection and screen, based on G418 antibiotic resistance and enhanced green fluorescent protein (EGFP), respectively, by placing the two marker genes KANMX and EGFP under the control of bidirectional GAL1p and GAL10p promoters;The bidirectional promoters GAL1p/GAL10p and HXT7t terminator were amplified from the yeast genome" "cerevisiae strain, VEG16 or VEG20 were transformed with mutant libraries of pVEG8 using established protocols69 and recovered for 6‰Ûäh in 1.2‰Ûäml of YP supplemented with 2% of sucrose and xylose before plating on the agar plates with same medium, supplemented with 100‰Ûäë_g/ml of G418 sulfate;coli NEB5ë± was used to transform the ligated mixture to create all the plasmids described using MES transformation except for mutant libraries, which were created by electroporating the ligation mixture;Random mutagenesis libraries were created by error prone PCR with 0.3‰Ûäng/ë_l of template plasmid, 0.2‰ÛämM dATP, 0.2‰ÛämM dGTP, 1‰ÛämM dCTP, 1‰ÛämM dTTP, 5‰ÛämM MgCl2, MnCl2 (0.05‰ÛämM for mutagenesis on the entire protein and 0.3‰ÛämM for mutations on the loops), 0.05‰ÛäU/ml Taq DNA polymerase, and 0.4‰ÛämM of the forward and reverse primers;Five transformants were randomly chosen and their gene sequenced to determine the error rate of the library;RNA extraction as well as library preparation and sequencing were outsourced to Genewiz, Inc;The obtained gene count data was normalized based on library size, converted to cpm (counts per million) using edgeR package;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "E.Z.N.A.å¨ Plasmid Mini Kit I, PCR Purification and Gel Extraction Kits were obtained from Omegabiotek (Norcross, GA)" http://dx.doi.org/10.1038/s41467-018-03645-7 TRUE
SAMN11998619 Illumina HiSeq 2500 sequencing; GSM3863173: WA1 Euploid Wild-type Rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM3863173: WA1 Euploid Wild-type Rep1; Saccharomyces cerevisiae; RNA-Seq 9/1/20 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR9257162 SAMN11998619 GSM3863173 WA1 Euploid Wild-type Rep1 NA Saccharomyces cerevisiae PRJNA548066 GSE132425 The Genetic Basis of Aneuploidy Tolerance in Wild Yeast ftp.sra.ebi.ac.uk/vol1/fastq/SRR925/002/SRR9257162/SRR9257162_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR925/002/SRR9257162/SRR9257162_2.fastq.gz 28 TRUE "Hose J et al., \The genetic basis of aneuploidy tolerance in wild yeast.\"", Elife, 2020 Jan 7;9""" NA "To distinguish these models, we transformed YPS1009 strains with a barcoded, high-copy gene over-expression library and measured relative fitness costs after 5 generations of growth (see MaterialsåÊandåÊmethods);Both the euploid and aneuploid ssd1ëÓ mutants were highly sensitive to the library (Figure 2D): genes that were deleterious in wild type were toxic in the mutant, while many genes with neutral effect in parental strains were deleterious in the absence of SSD1;Nonetheless, both the euploid and aneuploid ssd1ëÓ mutants are highly sensitive to the 2-micron overexpression library.;Pooled genomic DNA was sequenced using NEBNext Ultra DNA Library Prep Kit for Illumina on an Illumina HiSeq 2000 to an average of 20M 100 bp reads per pool;The suite of YPS1009_Chr12 strains (AGY731, AGY735, AGY1503, AGY1517) was transformed with Moby 2.0 high-copy expression library (Magtanong et al., 2011) and an aliquot removed as the starting pool;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Pooled genomic DNA was sequenced using NEBNext Ultra DNA Library Prep Kit for Illumina on an Illumina HiSeq 2000 to an average of 20M 100 bp reads per pool;Organelle-enriched and -depleted fractions were generated for euploid (AGY1446) and aneuploid YPS1009_Ch12 SSD1-GFP (AGY1447) and untagged SSD1 cells as a control, using Mitochondrial Yeast Isolation Kit (Abcam, Cambridge, United Kingdom) according to manufacturer protocol with slight modifications to minimize protein degradation" https://doi.org/10.7554/eLife.52063 TRUE
SAMN09216669 Illumina HiSeq 2000 sequencing; GSM3144853: rpb1-1/caf1_0; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM3144853: rpb1-1/caf1_0; Saccharomyces cerevisiae; RNA-Seq 26/6/18 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR7174202 SAMN09216669 GSM3144853 rpb1-1/caf1_0 NA Saccharomyces cerevisiae PRJNA471740 GSE114560 Determination of global decay rates of yeast transcriptome and identification of factors impact mRNA stability ftp.sra.ebi.ac.uk/vol1/fastq/SRR717/002/SRR7174202/SRR7174202_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR717/002/SRR7174202/SRR7174202_2.fastq.gz 10 TRUE "Webster MW et al., \mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.\"", Mol Cell, 2018 Jun 21;70(6):1089-1100.e8""" Stranded "Hybrid oligonucleotides were synthesized (IDT) with the RNA of interest (A30, or N20An, Table S2) at the 5‰Û_ end followed by single-stranded DNA complementary in sequence to the fluorescently labeled oligonucleotide on the chip;Libraries were prepared by using Illumina TruSeq Stranded Total RNA and mRNA library prep kits, quantified by an Agilent Bioanalyzer and sequenced by using paired-end 100åÊbp reads with an index read on Illumina HiSeq2000" "This process is unidirectional, because once the tail is shortened, the RRM is unable to rebind" "Following heat inactivation of Pol II in rpb1-1 and rpb1-1/caf1ëÓ strains, cells were harvested at various time points, and then global mRNA decay analysis was performed by RNA sequencing (RNA-seq) on libraries from each time point;Libraries were prepared by using Illumina TruSeq Stranded Total RNA and mRNA library prep kits, quantified by an Agilent Bioanalyzer and sequenced by using paired-end 100åÊbp reads with an index read on Illumina HiSeq2000;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "This was cloned into pGEX-6P-2 plasmid using an In-Fusion HD Cloning Kit (Clontech) for overexpression as an N-terminal GST-fusion in E.åÊcoli BL21 Star (DE3) cells;DNA encoding Pab1 amino acids 80‰ÛÒ653 was amplified from S.åÊpombe cDNA using primers Pab1_res80_Fwd and Pab1_Rev (Table S2) and cloned into pGEX-6P-2 plasmid using an In-Fusion HD Cloning Kit (Clontech);PCR-based site-directed mutagenesis was performed with a Quikchange Lightning Multi Mutagenesis Kit (Agilent) to generate mutations in Pab1 (RRM1mut: Y83A, RRM2mut: F171A, RRM3mut: Y264A, RRM4mut: F367A);Libraries were prepared by using Illumina TruSeq Stranded Total RNA and mRNA library prep kits, quantified by an Agilent Bioanalyzer and sequenced by using paired-end 100åÊbp reads with an index read on Illumina HiSeq2000" https://doi.org/10.1016/j.molcel.2018.05.033 TRUE
SAMN11419553 Illumina HiSeq 4000 sequencing; GSM3723041: H1: wild type HSF1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 4000 sequencing; GSM3723041: H1: wild type HSF1; Saccharomyces cerevisiae; RNA-Seq 17/4/19 Illumina HiSeq 4000 ILLUMINA PAIRED cDNA SRR8902390 SAMN11419553 GSM3723041 H1: wild type HSF1 NA Saccharomyces cerevisiae PRJNA532914 GSE129832 Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast ftp.sra.ebi.ac.uk/vol1/fastq/SRR890/000/SRR8902390/SRR8902390_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR890/000/SRR8902390/SRR8902390_2.fastq.gz 15 TRUE "Peffer S et al., \Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast.\"", J Biol Chem, 2019 Aug 9;294(32):12191-12202""" NA "National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "For qRT-PCR assays, 1 ë_g of RNA was converted to cDNA using the iScript cDNA synthesis kit (Bio-Rad)" https://doi.org/10.1074/jbc.ra119.008822 TRUE
SAMN07972768 Illumina HiSeq 2500 sequencing; GSM2838605: WT1-untreated; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2838605: WT1-untreated; Saccharomyces cerevisiae; RNA-Seq 3/5/19 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR6251545 SAMN07972768 GSM2838605 WT1-untreated NA Saccharomyces cerevisiae PRJNA416996 GSE106478 Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (Structure-seq) ftp.sra.ebi.ac.uk/vol1/fastq/SRR625/005/SRR6251545/SRR6251545_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR625/005/SRR6251545/SRR6251545_2.fastq.gz 10 TRUE "Lai YH et al., \Genome-Wide Discovery of DEAD-Box RNA Helicase Targets Reveals RNA Structural Remodeling in Transcription Termination.\"", Genetics, 2019 May;212(1):153-174""" NA "A single-strand DNA linker (Table 6) was ligated to the cDNA 3‰Û_ ends using CircLigase I (Epicentre) as described (Ding et al;Ignoring genes with sequence overlaps with at least one other gene on the same strand, we retained 4681 mRNAs and 77 snoRNAs for differential DMS reactivity analysis;This is likely to be true for NNS binding as both Nrd1 and Nab3 recognize RNA motifs in the context of single-strand RNA (Singh et al;This is likely to be true for NNS binding as both Nrd1 and Nab3 recognize RNA motifs in the context of single-strand RNA (Singh et al" "For this analysis, only transcripts without downstream overlapping genes in the sense direction and with over five read counts were considered (3428 mRNAs);Only transcripts without overlapping genes within 150 bp downstream in the sense direction were considered in the analysis" "Bolded letters are barcodes for different libraries.;All the samples were analyzed by Agilent Bioanalyzer to determine the size distribution of the library;Transcripts that had fewer than five counts in each library were filtered from the analysis;Sequencing libraries were prepared from the input and immunoprecipitated DNAs using NEXTflex ChIP-Seq Kit (BIOO Scientific, Austin, TX) according the manufacturer‰Ûªs instructions;All the libraries were analyzed by Agilent Bioanalyzer to determine the size distribution;The number of cross-linking counts in each bin was then normalized to the library size of each replicate and to the expression level to obtain reads per kilobase of transcript per million mapped reads (RPKM units) of each transcript based on the Structure-seq data [wild type, no dimethyl sulfate (DMS) treatment];After elution, the library was ethanol precipitated and resuspended in water;All the samples were analyzed by Agilent Bioanalyzer to determine the size distribution of the library;A total of 10 libraries, including three replicates of the wild type and two replicates of dbp2‰ö , with or without DMS treatment, were sequenced on the Illumina HiSeq 2500 platform for 2ÌÑ 100 bp paired-end cycle run.;Bolded letters are barcodes for multiple libraries.;For each mRNA, reads from untreated (no DMS) Structure-seq libraries mapped to the ORF or 150 nt downstream of the 3‰Û_ UTR (referred as extended region below) were counted using the summarizeOverlaps function (IntersectionNotEmpty mode) in the Bioconductor package ‰ÛÏGenomicAlignments‰Û (version 1.8.4);Only transcripts meeting the following conditions were analyzed: the extended region does not overlap with the downstream transcript, with >0.97 counts per million, and detected in at least two libraries;Input and immunoprecipitation libraries were prepared from three biological replicates of both wild-type and dbp2‰ö cells and resulted in an average of ‰ö_1.8 million reads per replicate;Methylated nucleotides were detected as RT ‰ÛÏstops,‰Û after library construction and RNA-seq, and then translated into nucleotide-level reactivities to DMS and a prediction of increased or decreased protection between wild type and dbp2‰ö (see Materials and Methods);Chang Laboratory) at Stanford University for providing the iCLIP-seq protocol, and Yiliang Ding (John Innes Centre) for help in construction of Structure-seq libraries;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Sequencing libraries were prepared from the input and immunoprecipitated DNAs using NEXTflex ChIP-Seq Kit (BIOO Scientific, Austin, TX) according the manufacturer‰Ûªs instructions" https://doi.org/10.1534/genetics.119.302058 TRUE
SAMN07711131 Illumina HiSeq 2500 sequencing; GSM2795704: bin HI; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2795704: bin HI; Saccharomyces cerevisiae; RNA-Seq 2/1/19 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR6108614 SAMN07711131 GSM2795704 bin HI NA Saccharomyces cerevisiae PRJNA412397 GSE104343 Single cell functional genomics reveals the importance of mitochondria in cell-to-cell phenotypic variation ftp.sra.ebi.ac.uk/vol1/fastq/SRR610/004/SRR6108614/SRR6108614_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR610/004/SRR6108614/SRR6108614_2.fastq.gz 16 TRUE "Dhar R et al., \Single cell functional genomics reveals the importance of mitochondria in cell-to-cell phenotypic variation.\"", Elife, 2019 Jan 14;8""" NA These results would be consistent with switching only in the direction of becoming petite;But the authors argue that switching can go in both directions because sorted bins could repopulate the full distributions of TMRE staining and growth;These results would be consistent with switching only in the direction of becoming petite;But the authors argue that switching can go in both directions because sorted bins could repopulate the full distributions of TMRE staining and growth "National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " Total RNA was isolated using MasterPure yeast RNA isolation kit (Epicentre) following manufacturer‰Ûªs protocol;Isolated total RNA (using MasterPure yeast RNA isolation kit (Epicentre)) was checked and quantified using bioanalyzer https://doi.org/10.7554/eLife.38904 TRUE
SAMN03333498 "Illumina HiSeq 2000 sequencing; GSM1604398: WT Young (S2Y, Exp 7); Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2000 sequencing; GSM1604398: WT Young (S2Y, Exp 7); Saccharomyces cerevisiae; RNA-Seq" 3/11/15 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR1793967 SAMN03333498 GSM1604398 "WT Young (S2Y, Exp 7)" NA Saccharomyces cerevisiae PRJNA274977 GSE65764 H3K36 methylation promotes longevity by enhancing transcriptional fidelity [Yeast RNA-Seq] ftp.sra.ebi.ac.uk/vol1/fastq/SRR179/007/SRR1793967/SRR1793967_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR179/007/SRR1793967/SRR1793967_2.fastq.gz 24 TRUE "Sen P et al., \H3K36 methylation promotes longevity by enhancing transcriptional fidelity.\"", Genes Dev, 2015 Jul 1;29(13):1362-76""" NA We thus prepared strand-specific libraries from polyA-selected RNA from young and old cells (SHORT RNA LIBRARY) "In this study, we report the results of a population-based high-throughput life span screen using a large-scale histone H3/H4 mutant library and identify H3K36 as an important residue modulating life span.;Thanks to the tractability of yeast genetics, systematic histone mutant libraries have been made available (Dai et al;We used the histone H3/H4 systematic mutant library developed in the Boeke laboratory (Dai et al;First, all mutations in this library are integrated into the yeast genome; therefore, complications caused by variations in natural loss rates of the histone plasmid during aging are avoided;Second, each mutant carries two unique barcodes that can be used for identification and quantification; hence, the library is compatible with high-throughput screening;Briefly, a pool of all mutants from the library was labeled with biotin, aged, and sorted multiple times to purify young and old fractions;Although seemingly lower than expected, this is a success rate comparable with a previously published yeast chronological life span screen using the same histone mutant library (Matecic et al;We thus prepared strand-specific libraries from polyA-selected RNA from young and old cells;This high-throughput approach, when combined with various yeast genetic libraries, can expedite mechanistic studies of aging, provided that life span assays can be validated by other established approaches.;All strains from the Boeke histone H3/H4 mutant library were pooled together with additional uniquely barcoded wild-type, sir2ëÓ, and SIR2-OE strains;To prepare next-generation sequencing libraries, primers U1h-F1 (CCCTACACGACGCTCTTCCGATCTATGTCCACGAGGTCTCT) and U2h-R1 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGACCTGCAGCGTA) were used to amplify UPTAG, and primers D1h-F1 (CCCTACACGACGCTCTTCCGATCTCCCAGCTCGAATTCATC) and D2h-R1 (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGGTGTCGGTCTCGTAG) were used for DNTAG;All next-generation sequencing libraries were indexed and sequenced in 1/10 of a lane in HiSeq 2000 (Illumina);To generate libraries for RNA-seq, mRNA was purified from 5 åµg of total RNA using the Dynabeads mRNA Direct kit (Ambion);Final size selection of the amplified library was done using 6% polyacrylamide gels;For the sake of consistency, RNA-seq libraries from worms were prepared using the same protocol;The quantity and quality of the libraries were assessed by BioAnalyzer (Agilent) and qPCR (Kapa Biosystems);Eight multiplexed libraries were pooled and sequenced on a single lane on the Illumina HiSeq or NextSeq 500 platform.;To ensure that we were getting H3K4me3 and H3K36me3 enrichment at 5‰Û_ ends and 3‰Û_ ends, respectively, ChIP-qPCR validation was performed with 5‰Û_ and 3‰Û_ end primer sets over long genes before library preparation for ChIP-seq;For ChIP-seq, 1 ng of DNA from immunoprecipitation and input was used to prepare libraries using the Microplex library preparation kit (Diagenode);Twelve multiplexed libraries were sequenced in a single lane on the Illumina HiSeq or NextSeq 500 platform.;We thank Jerry Workman for the strains YBL619, YBL634, and YBL694; the University of Pennsylvania Next-Generation Sequencing Core (NGSC) for sequencing our ChIP-seq and RNA-seq libraries; and members of the Berger laboratory for critical reading of the manuscript;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Total RNA was extracted from frozen cell pellets using the Qiagen RNeasy kit following the instructions, except that after resuspending in QIAzol lysis reagent, cells were subjected to bead beating;For qPCR, 1 åµg of total RNA was converted to cDNA using the high-capacity RNA to cDNA kit (Applied Biosystems);Northern blotting was performed using a NorthernMax kit (Life Technologies);Briefly, 10 åµg of total RNA was subjected to polyA selection using the Dynabeads mRNA Direct kit (Ambion) and loaded on a 1% formaldehyde agarose gel, blotted on a positively charged nylon membrane (Hybond N+, GE Healthcare), UV cross-linked, and then probed with radiolabeled PCR products designed against the 5‰Û_ end of STE11 and the 3‰Û_ end of STE11 and ACT1.;Total RNA was then extracted and purified with miRNeasy minikit (Qiagen).;To generate libraries for RNA-seq, mRNA was purified from 5 åµg of total RNA using the Dynabeads mRNA Direct kit (Ambion);For ChIP-seq, 1 ng of DNA from immunoprecipitation and input was used to prepare libraries using the Microplex library preparation kit (Diagenode)" https://doi.org/10.1101/gad.263707.115 TRUE
SAMN04453220 Illumina HiSeq 2500 sequencing; GSM2053504: WT_40min_1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2053504: WT_40min_1; Saccharomyces cerevisiae; RNA-Seq 14/2/19 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR3140907 SAMN04453220 GSM2053504 WT_40min_1 NA Saccharomyces cerevisiae PRJNA310655 GSE77511 The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast ftp.sra.ebi.ac.uk/vol1/fastq/SRR314/007/SRR3140907/SRR3140907_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR314/007/SRR3140907/SRR3140907_2.fastq.gz 12 TRUE "Xie B et al., \The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast.\"", RNA Biol, 2019 Jun;16(6):727-741""" Stranded (Truseq Stranded) "5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs;Such cases include enzymes important for nucleotide excision repair (RAD4), double strand break repair (PRI2), recombination (SMC6), mismatch repair (PMS1), and DNA-damage induced transcription (DUN1);Top (+) and bottom (-) DNA strands and a scale are given to the right;(e) A color-coded graph plots the 19‰ÛÒ23 base small RNA read densities (tag/nucleotide, y-axis) against top (+) and bottom (-) strands of the genome (y-axis);Intriguingly, the group of s/a loci includes the SWI5 paralog ACE2, and its overlapping antisense lncRNAs CUT259 and SUT2107 that show opposing peak expression patterns both in RNA-Sequencing and stranded RT-PCR experiments (Figure 8A-C);(E) A color-coded graph plots the 19‰ÛÒ23 base small RNA read densities (tag/nucleotide, y-axis) against top (+) and bottom (-) strands of the genome (y-axis) like in Figure 7F;The ACE2/SUT2107 double-stranded form covers almost the entire coding region of ACE2, and is associated with diminished Ace2 protein levels in 5-FU and rrp6‰ö samples;We next prepared strand-specific RNA-Sequencing libraries starting from 125 ng of rRNA-depleted RNA using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina, USA), as described [58];Before carrying out stranded RNA-Sequencing, we controlled total RNA and cDNA quality using the RNA 6000 Pico kit, and High Sensitivity DNA chips, respectively, and a BioAnalyzer 2100 (Agilent, France).;Strand-specific RT-PCR assays were carried out using the SuperScript II kit (ThermoScientific, USA);2åµg of RNA was treated with DNaseI (Ambion, USA), and reverse transcribed with reverse primers (shown in Table 2), in the presence of Actinomycin D to prevent second strand synthesis" "However, a growing body of evidence indicates that many of them are not a dispensable byproduct of bi-directional transcription, but regulate gene expression via promoter interference and sense/antisense (s/a) interference of transcription in both fission- and budding yeast [25‰ÛÒ31]." "We next prepared strand-specific RNA-Sequencing libraries starting from 125 ng of rRNA-depleted RNA using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina, USA), as described [58];We employed a HiSeq2500 sequencer (Illumina) to carry out paired-end sequencing of the libraries (2 x 50 nucleotides);Such a filtering step is recommended for microarray and RNA-Sequencing based profiling experiments, since genes with very low counts across libraries do not provide robust evidence for differential expression [83,84];The library size was recomputed after this filtering step and counts were normalized using the TMM approach [85], and the differential analysis was performed with EdgeR [86,87];National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Ribosomal RNA was removed using the RiboMinus Eukaryote System v2 kit (ThermoFisher, France);We next prepared strand-specific RNA-Sequencing libraries starting from 125 ng of rRNA-depleted RNA using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina, USA), as described [58];Before carrying out stranded RNA-Sequencing, we controlled total RNA and cDNA quality using the RNA 6000 Pico kit, and High Sensitivity DNA chips, respectively, and a BioAnalyzer 2100 (Agilent, France).;Finally, precipitated RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (ThermoScientific, USA) and amplified using Taq Polymerase (Qiagen, France) at 60å¼C for 30 cycles using the primer pair for ACE2 (Table 2).;Strand-specific RT-PCR assays were carried out using the SuperScript II kit (ThermoScientific, USA);Briefly, RNA was extracted using the hot phenol method, followed by DNase I treatment with TURBO DNA-free Kit (Ambion, USA);RT-PCR reactions were carried out using 2åÊåµg of RNA reverse transcribed with Reverse Transcriptase (High Capacity cDNA Reverse Transcription kit; Life Technologies, USA) and amplified using Taq Polymerase (Qiagen, France) at 60å¼C for 28 cycles;Protein signals were revealed using the ECL-Plus Chemiluminescence kit (GE Healthcare, USA) and the ChemiDoc XRS system (Bio-Rad, USA)" http://www.tandfonline.com/doi/full/10.1080/15476286.2019.1581596 TRUE
SAMN06469688 Illumina HiSeq 2500 sequencing; GSM2519962: WT_Rap_repeat1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2519962: WT_Rap_repeat1; Saccharomyces cerevisiae; RNA-Seq 10/7/17 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR5309371 SAMN06469688 GSM2519962 WT_Rap_repeat1 NA Saccharomyces cerevisiae PRJNA377731 GSE95633 MINC Regulates Pervasive Transcription in Yeast and Mammals ftp.sra.ebi.ac.uk/vol1/fastq/SRR530/001/SRR5309371/SRR5309371_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR530/001/SRR5309371/SRR5309371_2.fastq.gz 25 TRUE "Xue Y et al., \Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.\"", Mol Cell, 2017 Aug 17;67(4):594-607.e4""" Stranded "To determine the transcriptome consequences of depleting MINC, we performed strand-specific RNA sequencing in rapamycin-treated AA strains of MINC components and plotted the results as heat maps representing the fold change versus parental strains (blue is downregulated and red is upregulated by AA);(C) Same as A but for sense strand transcription (C4, 652 genes; C5, 1002 genes; C6, 1092 genes);The data on the sense strand revealed additional insights into the silencing role of MINC;(C) Same as A but for sense strand transcription;For yeast, libraries of mRNA were prepared with a KAPA stranded mRNA-Seq kit;For mouse total and nascent RNA, libraries were prepared with KAPA stranded RNA-Seq with RiboErase kit;Sequenced reads were aligned as above using tophat2.2.1 with option ‰ÛÒg 1 ‰ÛÒN 2 --no-mixed --no-discordant --library-type=fr-firststrand (Langmead et al., 2009);For strand specific reads, the mapped plus or minus strand transcripts were separately plotted against genes on the minus or plus strand for metagene analysis" "One RNA initiates in the forward direction into the gene;The other initiates in the antisense or reverse direction;Y axis shows the normalized read counts and the arrows indicate the direction of transcripts;Known 5‰Û_ Cap sites and directions are indicated as arrows in TSSs row;Y axis shows the normalized read counts and arrows indicate the direction of transcripts;Known 5‰Û_ Cap sites and direction of pervasive RNA are indicated in the TSSs row;Shaded areas represent pervasive transcripts in the antisense direction upstream of the TSS (Suco) and sense direction downstream of the TTS (Mef2d);Natural promoters employ several recognition sequences and multiple mechanisms, including evolutionary selection, to ensure that TBP, in the context of TFIID, recognizes physiological promoters with high affinity and a specific directionality (Kadonaga, 2012)" "Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 or 4000 platform for 50 bp single end reads;Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 or 4000 platform for 50 bp single end reads;RNAs were treated with DNase before library preparation;For yeast, libraries of mRNA were prepared with a KAPA stranded mRNA-Seq kit;For mouse total and nascent RNA, libraries were prepared with KAPA stranded RNA-Seq with RiboErase kit;Libraries were sequenced on the Illumina HiSeq 2500 for 50 bases in paired end read mode;Sequenced reads were aligned as above using tophat2.2.1 with option ‰ÛÒg 1 ‰ÛÒN 2 --no-mixed --no-discordant --library-type=fr-firststrand (Langmead et al., 2009);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "cerevisiae subtelomeric heterochromatin displayed a bi-stable state where embedded genes could become active even when bound by Sir proteins (Kitada et al., 2012);Briefly, 5 micrograms of a 602-bp template encompassing G5E4T was assembled into chromatin as described (Kitada et al., 2012), immobilized onto paramagnetic beads and incubated with template-saturating amounts (~10 micrograms) of Sir3 and Sir2/4 in 2.5 ml of binding buffer (100 mM KOAc, 20 mM Hepes, pH 7.6, 1 mM EDTA and 10% glycerol) for 1h at 30å¡C;The supernatant from sonicated lysates were precleared with Protein A/G beads and ChIP was performed as described (Kitada et al., 2012) using commercial antibodies: anti-Myc (Abcam, ab32, GR255064-2), anti-Arp5 (Abcam, ab12099), anti-H3K79me3 (Grunstein lab, 644) and 0.5% BSA (w/v) pre-blocked Protein A/G beads;Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 or 4000 platform for 50 bp single end reads;Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 or 4000 platform for 50 bp single end reads;The extracted RNA samples were treated with DNase I (Ambion TURBO DNA-free Kit) and further purified with Trizol regents (Ambion);For yeast, libraries of mRNA were prepared with a KAPA stranded mRNA-Seq kit;For mouse total and nascent RNA, libraries were prepared with KAPA stranded RNA-Seq with RiboErase kit" https://doi.org/10.1016/j.molcel.2017.06.029 TRUE
SAMN03075996 "Illumina HiSeq 2500 sequencing; GSM1510423: pho85_DaMP, high Pi, 0 min; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2500 sequencing; GSM1510423: pho85_DaMP, high Pi, 0 min; Saccharomyces cerevisiae; RNA-Seq" 21/3/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR1583766 SAMN03075996 GSM1510423 "pho85_DaMP, high Pi, 0 min" NA Saccharomyces cerevisiae PRJNA261792 GSE61663 Transcription profile of pho85 damp strain during growth in no phosphate medium ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/006/SRR1583766/SRR1583766_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/006/SRR1583766/SRR1583766_2.fastq.gz 25 TRUE "Vardi N et al., \Sequential feedback induction stabilizes the phosphate starvation response in budding yeast.\"", Cell Rep, 2014 Nov 6;9(3):1122-34""" NA NA NA NA NA https://doi.org/10.1016/j.celrep.2014.10.002 FALSE
SAMN03098164 Illumina HiSeq 2000 sequencing; GSM1508098: RNA seq of hybrid A; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM1508098: RNA seq of hybrid A; Saccharomyces cerevisiae; RNA-Seq 6/2/15 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR1605749 SAMN03098164 GSM1508098 RNA seq of hybrid A NA Saccharomyces cerevisiae PRJNA263283 GSE61553 Transcription profiling of a S96xSK1 yeast cross and its pool of spores. ftp.sra.ebi.ac.uk/vol1/fastq/SRR160/009/SRR1605749/SRR1605749_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR160/009/SRR1605749/SRR1605749_2.fastq.gz 12 TRUE "Bader DM et al., \Negative feedback buffers effects of regulatory variants.\"", Mol Syst Biol, 2015 Jan 29;11(1):785""" Stranded "Strand-specific total RNA-Seq libraries were prepared as described in Wilkening etåÊal (2013) which is a modified protocol of Parkhomchuk etåÊal (2009);Of 8åÊë_l 5ÌÑ First strand buffer (Invitrogen), 4åÊë_l DTT 0.1åÊM, 0.5åÊë_l actinomycin D (1.25åÊmg/ml) and 0.5åÊë_l RNasin plus RNase inhibitor (Promega) were added to each sample and the samples were then incubated at C for 2åÊmin;After cleanup, the 2nd strand cDNA synthesis was done with dUTPs instead of dTTPs;The dUTPs of the second strand were hydrolyzed by incubating the samples at C for 30åÊmin with 5 units of UDG in UDG reaction buffer (NEB)" "For the 984 cis genes, allelic expression imbalances typically agreed in direction, but were weaker for the pool of spores compared to the hybrid (Fig‰ÛÜ(Fig2A,2A, mass of the data subdiagonal);The SK1 gene annotation was generated via bidirectional best hits: Using the coordinates from Xu and colleagues, we extracted the S96 gene sequences from the S288c genome version R64 of the Saccharomyces Genome Database (Cherry etåÊal, 2012)" "Strand-specific total RNA-Seq libraries were prepared as described in Wilkening etåÊal (2013) which is a modified protocol of Parkhomchuk etåÊal (2009);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " Total RNA was isolated by a standard hot phenol method followed by DNase treatment using Turbo DNA-free kit (Ambion) https://doi.org/10.15252/msb.20145844 TRUE
SAMN06321071 Illumina HiSeq 2000 sequencing; GSM2483165: fkh2ëÓ RNA-Seq rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM2483165: fkh2ëÓ RNA-Seq rep1; Saccharomyces cerevisiae; RNA-Seq 13/3/17 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR5245224 SAMN06321071 GSM2483165 fkh2ëÓ RNA-Seq rep1 NA Saccharomyces cerevisiae PRJNA374346 GSE94795 Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae (HTS) ftp.sra.ebi.ac.uk/vol1/fastq/SRR524/004/SRR5245224/SRR5245224_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR524/004/SRR5245224/SRR5245224_2.fastq.gz 19 TRUE "Ostrow AZ et al., \Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.\"", Proc Natl Acad Sci U S A, 2017 Mar 21;114(12):E2411-E2419""" UNKNOWN cDNA libraries were created by using SuperScript III First-Strand Synthesis (ThermoFisher 18080051) with oligo dT primers followed by NEBNext Second Strand Synthesis (NEB E6111S) "Similarly, hundreds of replication origins coalesce into foci containing several origins each, which become bidirectional replisomes that remain colocalized as DNA is spooled through during replication (reviewed in ref" "cDNA libraries were created by using SuperScript III First-Strand Synthesis (ThermoFisher 18080051) with oligo dT primers followed by NEBNext Second Strand Synthesis (NEB E6111S);cDNA libraries were prepared for sequencing and analyzed as described for BrdU-IP-Seq;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "The dsm mutations were introduced into the above plasmids by using QuikChange Lightning Mutagenesis Kit (Agilent Technologies), yielding pGAL-fkh1-dsm-MYC9, p405-fkh1-dsm, and p405-fkh2-dsm;For RNA-seq analysis, total RNA was isolated from 10-mL cultures grown in YEPD to OD600 ‰ö_ 1 by using Ribopure Yeast Kit (ThermoFisher AM1926)" https://doi.org/10.1073/pnas.1612422114 TRUE
SAMN03076081 "Illumina HiSeq 2500 sequencing; GSM1510475: pho90_OX, no Pi, 30min; Saccharomyces cerevisiae; RNA-Seq" "Illumina HiSeq 2500 sequencing; GSM1510475: pho90_OX, no Pi, 30min; Saccharomyces cerevisiae; RNA-Seq" 21/3/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR1583818 SAMN03076081 GSM1510475 "pho90_OX, no Pi, 30min" NA Saccharomyces cerevisiae PRJNA261795 GSE61665 Transcription profile of pho90_OX strain during growth in no phosphate medium ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/008/SRR1583818/SRR1583818_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR158/008/SRR1583818/SRR1583818_2.fastq.gz 23 TRUE "Vardi N et al., \Sequential feedback induction stabilizes the phosphate starvation response in budding yeast.\"", Cell Rep, 2014 Nov 6;9(3):1122-34""" NA NA NA NA NA https://doi.org/10.1016/j.celrep.2014.10.002 FALSE
SAMN04216014 Illumina HiSeq 2500 sequencing; GSM1918909: SC_D_rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM1918909: SC_D_rep1; Saccharomyces cerevisiae; RNA-Seq 16/12/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR2817418 SAMN04216014 GSM1918909 SC_D_rep1 NA Saccharomyces cerevisiae PRJNA299815 GSE74361 Inhibiting Fungal Multidrug Resistance by Disrupting an Activator-Mediator Interaction With the Small Molecule iKIX1 ftp.sra.ebi.ac.uk/vol1/fastq/SRR281/008/SRR2817418/SRR2817418_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR281/008/SRR2817418/SRR2817418_2.fastq.gz 12 TRUE "Nishikawa JL et al., \Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.\"", Nature, 2016 Feb 25;530(7591):485-9""" Stranded (NEBNext Ultra Directional RNA Library Prep Kit) "Left: Table of compound libraries that were screened using a fluorescence polarization assay at the Institute of Chemistry & Cell Biology (ICCB) facility at Harvard Medical School.;We acknowledge the ICCB-Longwood Screening Facility at Harvard Medical School for assistance with the high-throughput screens and access to the compound libraries, and the MGH Next Gen sequencing core for RNA-Seq library construction;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " https://doi.org/10.1038/nature16963 TRUE
SAMN05990905 Illumina HiSeq 2000 sequencing; GSM2385251: SPT6 WT_polyA_exp1_rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM2385251: SPT6 WT_polyA_exp1_rep1; Saccharomyces cerevisiae; RNA-Seq 4/4/17 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR5000465 SAMN05990905 GSM2385251 SPT6 WT_polyA_exp1_rep1 NA Saccharomyces cerevisiae PRJNA352693 GSE89601 Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighbouring genes ftp.sra.ebi.ac.uk/vol1/fastq/SRR500/005/SRR5000465/SRR5000465_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR500/005/SRR5000465/SRR5000465_2.fastq.gz 12 TRUE "Uwimana N et al., \Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighboring genes.\"", Nucleic Acids Res, 2017 Jun 20;45(11):6417-6426""" Stranded "Strand-specific RNA-Seq libraries were prepared using the KAPA stranded RNA-Seq library preparation kit (KAPA Biosystems) prior to paired-end sequencing on an Illumina Hi-Seq2000;Cryptic transcripts running on the antisense strand were detected using StringTie (26);We next removed all the transcripts that overlapped with known annotations on the same strand to keep de novo antisense cryptic transcripts;The resulting scores were used to look at the motif distribution relative to genes on both sense and antisense strands using VAP (28,29).;For each gene, we calculated the probability of a termination event in the ‰ÛÒ250 and +250 bp region around their TTS on the antisense strand, using both the f score and the z score;Putative terminators (500 bp fragments) were then cloned downstream of this 5ëã UTR and termination was monitored by northern blotting using a single stranded RNA probe corresponding to the YNR051C 5ëã UTR;n = 5695 genes, which exclude dubious and overlapping genes on the same strand;Contrary to intragenic sense cryptic transcripts, antisense cryptic transcripts are more easily identified since very little signal is detected on the antisense strand of genes in WT cells;We therefore used a standard assembler (26) to identify de novo transcripts overlapping annotated genes on the antisense strand (Figure ‰ÛÜ(Figure1C).1C);This number is most likely an underestimation of the full set of antisense cryptic transcripts because our algorithm (as would be the case for any RNA-Seq-based method) is not efficient at identifying antisense cryptic transcripts that invade the neighboring gene on the same strand (due to merging of the RNA-Seq signal);Interestingly, antisense cryptic transcripts are globally expressed at lower levels than those on the sense strand (Figure ‰ÛÜ(Figure2B).2B);It is not clear whether this is due to differences in transcription or RNA stability, although cryptic transcripts from both strands appear to be polyadenylated (see below);For technical reasons (see above), very few antisense cryptic transcripts were identified that invade the upstream gene on the same strand (opposite strand to the gene where the antisense cryptic transcript has initiated), making it difficult to evaluate where these transcripts terminate;These regions contain terminators, but on the opposite strand;This prompted us to investigate whether known DNA motifs involved in polyadenylation-dependent termination were present on both strands in terminator regions;In order to look for evidence of terminators on the antisense strand, we mapped the density of the ‰Û÷efficiency‰Ûª and ‰Û÷positioning‰Ûª motifs (the other motifs having poor information content on their own), relative to genes, on both strands;As expected, when looking on the sense strand, we found enrichment for both motifs at the 3ëã-end of annotated genes (Figure ‰ÛÜ(Figure3C3C andåÊD, blue);Surprisingly, however, both motifs were also enriched on the antisense strand (Figure ‰ÛÜ(Figure3C3C andåÊD, gold);RNA-Seq signal on the Watson (gray) and Crick (black) strands is shown;(C) Aggregate profile of the efficiency motif (UAUAUA, UACAUA, UAUGUA) enrichment on the sense (blue) and antisense (gold) strands over genes;(D) Aggregate profile of the positioning motif (AAUAAA, AAAAAA) enrichment on the sense (blue) and antisense (gold) strands over genes;We noticed, however, that the level of antisense cryptic transcripts is on average 1.6 (mean) fold less abundant than those on the sense strand (see Figure ‰ÛÜFigure2B),2B), suggesting that they may be terminating via another pathway leading to less stable transcripts;Among the eight terminators that were previously shown to be bidirectional (ARO4, TRP1, TRP4, ADH1, CYC1, GAL1, GAL7, GAL10), only one, ARO4, is facing a cryptic transcript coming from the antisense strand in spt6-1004 cells;RNA-Seq signal on the Watson (gray) and Crick (black) strand are shown;The green transcript (cryptic antisense) terminates at the terminator of Gene A, despite the fact that Gene A is on the other strand;Consistently, we found that DNA motifs characteristic of yeast terminators are enriched on both strands in the 3ëã-end of genes" "This finding led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from both directions;Interestingly, we found that antisense cryptic transcription often terminates at the terminator of the adjacent gene, thanks to the previously underestimated bidirectionality of most yeast terminators.;Gene terminators that are challenged by a cryptic transcript were classified as unidirectional or bidirectional as follows;Unidirectional terminators are defined as terminators that allow termination of their corresponding sense transcripts but are inefficient at terminating antisense transcription;Bidirectional terminators are defined as terminators which allow both sense and antisense transcription termination;Terminators were classified as bidirectional if they allow termination of antisense transcription within a 500 bp window around their annotated transcription termination sites (TTS);This method identified 579 bidirectional terminators;These are indication of terminators that are bidirectional but with weaker activity;To identify those weak bidirectional TTS, we used a probabilistic method similar to that used to identify cryptic transcripts;We thus selected bidirectional promoters having f and z scores values smaller than ‰ÛÒ94.47 and ‰ÛÒ11.35 respectively;Using this approach, we identified 97 weak bidirectional terminators;Unidirectional terminators were identified by selecting TTS that overlap an antisense cryptic transcript, but are located at least 250 bp away from the cryptic TTS;We found 150 unidirectional terminators using this criterion;See Supplementary Table S4 for the directionality call for each terminator evaluated.;Sense and antisense transcripts emerge from the same gene slightly more often than expected by chance but we often detect genes with a cryptic transcript running only in one direction (Figure ‰ÛÜ(Figure2C).2C);Alternatively, this may reflect imperfect mapping of some cTSS, especially in the sense direction;The data shown above implies that many, perhaps most, yeast terminators are functionally bidirectional;Terminator bidirectionality has been described anecdotally for a few yeast genes (35‰ÛÒ37), but its prevalence was never thoroughly investigated;Among the eight terminators that were previously shown to be bidirectional (ARO4, TRP1, TRP4, ADH1, CYC1, GAL1, GAL7, GAL10), only one, ARO4, is facing a cryptic transcript coming from the antisense strand in spt6-1004 cells;Satisfyingly, this cryptic transcript indeed terminates around the ARO4 terminator (not shown) demonstrating that our data can capture terminator bidirectionality;Because hundreds of antisense cryptic transcripts emerge in spt6-1004 cells, we reasoned that this could be used as an opportunity to classify terminators as uni- or bidirectional;From the 1616 antisense cryptic transcripts identified in spt6-1004 cells, we could predict the directionality of 826 terminators;Of those, 676 were classified as bidirectional and 150 as unidirectional (see Materials and Methods) (Figure ‰ÛÜ(Figure4A).4A);Figure ‰ÛÜFigure4B4B shows examples of bidirectional (left) and unidirectional (right) terminators identified in our analysis;Visual inspection of these putatively unidirectional terminators suggests that many of them are likely to be bidirectional but were not captured by our directionality prediction algorithm;These analyses predict that >80% of yeast promoters are functionally bidirectional.;Yeast terminators are mostly bidirectional;(A) Pie charts displaying the terminators that are challenged by antisense cryptic transcription (aCT; left) and the number of bidirectional, weak bidirectional and unidirectional terminators (right);(B) Genome-browser snapshots illustrating examples of bidirectional (left) and unidirectional (right) terminators;(D) RNA blot for the terminator assay testing terminators predicted to be bidirectional (s, terminator cloned in sense orientation; as, terminator cloned in antisense orientation);(E) Same as ‰Û÷D‰Ûª but for terminators predicted to be unidirectional;In order to challenge this prediction, we tested bidirectionality of a set of terminators from each group;When cloned in the inverted orientation (antisense), all terminators predicted to be bidirectional efficiently terminated transcription, confirming their bidirectionality (Figure ‰ÛÜ(Figure4D).4D);Among the terminators predicted to be unidirectional, some (YNL058C, HIS1 and GCN4) did not generate a robust signal for short transcripts when cloned in the reverse orientation (Figure ‰ÛÜ(Figure4E).4E);This is consistent with them being unidirectional terminators although we cannot exclude the possibility that short transcripts are generated but are unstable;The three other terminators predicted to be unidirectional (YGL101W, HMG2 and YJL136C), however, clearly behaved as bidirectional (Figure ‰ÛÜ(Figure4E),4E), suggesting that our predictions are overestimating the number of unidirectional terminators;Taken together, our RNA-Seq analyses and Northern blot experiments establish bidirectionality as a prevalent characteristic of most yeast terminators;Importantly, these analyses clearly show that terminator bidirectionality allows the termination of antisense cryptic transcription from invading neighboring genes (Figure ‰ÛÜ(Figure55).;This is evidence that the terminator of Gene A is bidirectional;cerevisiae are bidirectional;This prompted us to systematically predict the ability of yeast terminators to terminate transcription coming from the other direction;We therefore conclude that most yeast terminators are functionally bidirectional;Assuming that these polyadenylated transcripts are coupled to the termination of their associated polymerases, the bidirectional nature of yeast terminators would prevent/attenuate antisense transcription from invading the upstream gene when it is in tandem;cerevisiae are intrinsically bidirectional but divergent transcription is rapidly terminated via the Nrd1 pathway (39,40);In spt6-1004 cells, the very high abundance of these transcripts may saturate the capacity of the Nrd1 pathway so that we may be capturing the ‰Û÷escapees‰Ûª that terminate at the bidirectional terminators of the upstream gene as a backup mechanism." "Prior to library preparation, total RNA was either depleted for ribosomal RNA using the Ribo-zero Gold yeast kit (Epicentre-Illumina) or enriched for polyadenylated RNA using the NEBnext Poly(A) kit (New England Biolabs);Strand-specific RNA-Seq libraries were prepared using the KAPA stranded RNA-Seq library preparation kit (KAPA Biosystems) prior to paired-end sequencing on an Illumina Hi-Seq2000;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "Prior to library preparation, total RNA was either depleted for ribosomal RNA using the Ribo-zero Gold yeast kit (Epicentre-Illumina) or enriched for polyadenylated RNA using the NEBnext Poly(A) kit (New England Biolabs);Strand-specific RNA-Seq libraries were prepared using the KAPA stranded RNA-Seq library preparation kit (KAPA Biosystems) prior to paired-end sequencing on an Illumina Hi-Seq2000" https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkx242 TRUE
SAMN06688260 Illumina HiSeq 2000 sequencing; GSM2564140: Wildtype replicate 1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM2564140: Wildtype replicate 1; Saccharomyces cerevisiae; RNA-Seq 19/5/17 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR5417301 SAMN06688260 GSM2564140 Wildtype replicate 1 NA Saccharomyces cerevisiae PRJNA381711 GSE97416 The histone variant H2A.Z promotes efficient co-transcriptional splicing in S. cerevisiae ftp.sra.ebi.ac.uk/vol1/fastq/SRR541/001/SRR5417301/SRR5417301_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR541/001/SRR5417301/SRR5417301_2.fastq.gz 15 TRUE "Neves LT et al., \The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae.\"", Genes Dev, 2017 Apr 1;31(7):702-717""" Unstranded (Truseq v3) Two micrograms to 4 åµg of RNA was used to make cDNA using the Maxima first strand cDNA synthesis kit (Fermentas) "(B) Hierarchical clustering of H2A.Z-binding profiles of intron-containing non-RPGs around TSSs or splice sites, oriented gene-directionality;(C) Hierarchical clustering of H2A.Z-binding profiles of intron-containing RPGs around TSSs or splice sites, oriented gene-directionality" "RNA-seq libraries were prepared using an Illumina Truseq V3 kit and ribosomal RNA depletion;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " RNA-seq libraries were prepared using an Illumina Truseq V3 kit and ribosomal RNA depletion;Two micrograms to 4 åµg of RNA was used to make cDNA using the Maxima first strand cDNA synthesis kit (Fermentas);(2015) and converted to FastQ format using the NCBI Sequence Read Archive (SRA) toolkit;All samples were incubated with Proteinase K (Sigma) and RNase A (Ambion) followed by purification using a PCR product purification kit (Qiagen). https://doi.org/10.1101/gad.295188.116 TRUE
SAMN03001827 Illumina HiSeq 2000 sequencing; GSM1483710: BY_RM_0min_R1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 sequencing; GSM1483710: BY_RM_0min_R1; Saccharomyces cerevisiae; RNA-Seq 21/3/15 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR1555415 SAMN03001827 GSM1483710 BY_RM_0min_R1 NA Saccharomyces cerevisiae PRJNA258654 GSE60617 Heritable variation of mRNA decay rates in yeast ftp.sra.ebi.ac.uk/vol1/fastq/SRR155/005/SRR1555415/SRR1555415_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR155/005/SRR1555415/SRR1555415_2.fastq.gz 18 TRUE "Andrie JM et al., \Heritable variation of mRNA decay rates in yeast.\"", Genome Res, 2014 Dec;24(12):2000-10""" Unstranded (Truseq v2) "It is difficult to precisely estimate the proportion of genes with significant ASD that is in the opposite direction of steady-state expression levels because of differences in the statistical power of detecting ASE and ASD;Of these 34 genes, nine exhibit significant ASD, which is significantly more than we would expect by chance (Fisher‰Ûªs exact test, P-value = 5.16 ÌÑ 10‰öÕ3); however, only three of the nine genes show decay rate differences in the direction expected if allelic differences in mRNA decay rate were mediated by nonsense-mediated decay" "We used a TruSeq RNA Sample Prep v2 Kit (Illumina) to create a sequencing library from the total RNA collected for each decay rate time course time point from each replicate;We created barcoded sequencing libraries from the cDNA from each sample and, in an effort to minimize technical variation between the data acquired from different decay rate time points, all samples from all replicates were sequenced in the same lane on an Illumina HiSeq 2000 (50-bp paired-end reads).;We prepared a DNA sequencing library using a TruSeq DNA Sample Prep v2 Kit (Illumina);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "We used a TruSeq RNA Sample Prep v2 Kit (Illumina) to create a sequencing library from the total RNA collected for each decay rate time course time point from each replicate;Per the protocol for the kit, we isolated mRNA from the total RNA using two rounds of poly(A) selection, then fragmented the isolated mRNA into ‰ö_150 base pair (bp) fragments, and finally, used random hexamer primers to produce cDNA;We extracted DNA using a Genomic-tip 100/G Kit (Qiagen) and then concentrated the sample using a standard ethanol precipitation;We prepared a DNA sequencing library using a TruSeq DNA Sample Prep v2 Kit (Illumina);Per the protocol for the kit, we used a Covaris sonicator to shear the DNA into ‰ö_300- to 400-bp fragments, and, after ligating adaptors onto the DNA fragments, we additionally size-selected for 300- to 400-bp fragments by running the ligation products out on an agarose gel and gel-extracting the appropriate band" https://doi.org/10.1101/gr.175802.114 TRUE
SAMN01113244 Illumina HiSeq 2000 paired end sequencing; GSM985057: 3tfill_ypd_rep1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2000 paired end sequencing; GSM985057: 3tfill_ypd_rep1; Saccharomyces cerevisiae; RNA-Seq 11/11/13 Illumina HiSeq 2000 ILLUMINA PAIRED cDNA SRR539261 SAMN01113244 GSM985057 3tfill_ypd_rep1 NA Saccharomyces cerevisiae PRJNA172855 GSE40110 Saccharomyces cerevisiae 3' poly(A) site mapping ftp.sra.ebi.ac.uk/vol1/fastq/SRR539/SRR539261/SRR539261_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR539/SRR539261/SRR539261_2.fastq.gz 30 TRUE "Wilkening S et al., \An efficient method for genome-wide polyadenylation site mapping and RNA quantification.\"", Nucleic Acids Res, 2013 Mar 1;41(5):e65""" Stranded "Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner;After second strand synthesis, fragments are captured on beads and the barcoded adapter PE 2.0 (brown) is ligated to the bound fragment;Four microliters of 5ÌÑ first-strand buffer (Invitrogen), 2 åµl DTT 0.1 M, 0.32 åµl actinomycin D (1.25 mg/åµl) and 0.5 åµl RNasin plus RNase inhibitor (Promega) were added to each sample, and samples were incubated at 42å¡C for 2 min to minimize possible mispriming;For producing the second cDNA strand, 40 åµl of sample was mixed with 5 åµl of 10ÌÑ DNA polymerase buffer (Fermentas), 2.5 åµl of dNTPs (10 mM), 0.5 åµl of RNaseH (5 U/åµl; NEB) and 2 åµl of DNA polymerase I (10 U/åµl; Fermentas);Twenty microliters of the double-stranded cDNA sample was bound to the 20 åµl of Dynabeads by mixing them for 15 min at 25å¡C;In addition to a strand-specific RNA-Seq protocol (26), with very few reads that map poly(A) sites (Figure 2), we evaluated four poly(A)-mapping methods, including one RNA-Seq following enrichment of poly(A)-containing RNA fragments (as in (13)), one which sequences into the poly(A) tail called ‰Û÷3‰Û_Internal‰Ûª (27), similar to (14,16), one which cleaves off the poly(A) tail called ‰Û÷3‰Û_BpmI‰Ûª (similar principle to Jan et al;To benchmark the RNA quantification efficiency of our approach, we compared the results obtained with our 3‰Û_T-fill method to those from standard, strand-specific RNA-Seq;Poly(A) site positions on sense (upper panels in black) and antisense strands (lower panels in gray) relative to annotated (a) transcription start sites (left), transcription termination sites (right), (b) ORF starts (left) and ORF ends (right);As only a very small fraction of reads obtained from a strand-specific RNA-Seq run contain the poly(A) site (Figure 2), several methods that aim to identify the poly(A) sites have been developed, yet to date no comparative analysis of these protocols has been performed" "Compared with protocols where sequencing proceeds from the 5‰Û_-direction into the poly(A) tail, this protocol offers several advantages: size selection is not critical, no trimming is necessary and both forward and reverse reads are informative" "300 bp libraries were size selected using e-Gel 2% SizeSelect (Invitrogen).;The final libraries were loaded into the cluster station (cBot, Illumina) and the priming buffer was exchanged for the T-fill solution: 101 åµl water, 20 åµl Phusion buffer HF (5ÌÑ) (NEB), 3 åµl dTTPs (10 mM), 0.8 åµl genomic DNA Sequencing primer V2 (100 åµM) (Illumina), 3 åµl non-hot start Phusion polymerase (2 U/åµl, NEB) and 2 åµl Taq polymerase E (5 U/åµl; Genaxxon);It avoids delicate RNA processing and is flexible in library size and read length;National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion) https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gks1249 TRUE
SAMN04880646 Illumina HiSeq 2500 sequencing; GSM2128928: wild type no stress [Sample_1_13462_CGATGT]; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM2128928: wild type no stress [Sample_1_13462_CGATGT]; Saccharomyces cerevisiae; RNA-Seq 6/11/16 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR3407195 SAMN04880646 GSM2128928 wild type no stress [Sample_1_13462_CGATGT] NA Saccharomyces cerevisiae PRJNA319164 GSE80512 Gene expression analysis using RNA Sequencing of the Saccharomyces cerevisiae HTA1-S121A strain upon thermo and osmotic stress. ftp.sra.ebi.ac.uk/vol1/fastq/SRR340/005/SRR3407195/SRR3407195_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR340/005/SRR3407195/SRR3407195_2.fastq.gz 18 TRUE "Studer RA et al., \Evolution of protein phosphorylation across 18 fungal species.\"", Science, 2016 Oct 14;354(6309):229-232""" Stranded (TruSeq Stranded) NA NA NA NA https://doi.org/10.1126/science.aaf2144 FALSE
SAMN11840754 Illumina HiSeq 4000 sequencing; GSM3814232: MA replicate1; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 4000 sequencing; GSM3814232: MA replicate1; Saccharomyces cerevisiae; RNA-Seq 25/5/19 Illumina HiSeq 4000 ILLUMINA PAIRED cDNA SRR9114348 SAMN11840754 GSM3814232 MA replicate1 NA Saccharomyces cerevisiae PRJNA544475 GSE131702 Transcriptome-wide expression profile of yeast under different carbon sources ftp.sra.ebi.ac.uk/vol1/fastq/SRR911/008/SRR9114348/SRR9114348_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR911/008/SRR9114348/SRR9114348_2.fastq.gz 22 TRUE "Wang Y et al., \Single-molecule long-read sequencing reveals the chromatin basis of gene expression.\"", Genome Res, 2019 Aug;29(8):1329-1342""" NA NA NA NA FALSE FALSE
SAMN13614527 Illumina HiSeq 2500 sequencing; GSM4222243: WT_4705_A; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM4222243: WT_4705_A; Saccharomyces cerevisiae; RNA-Seq 12/1/20 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR10720198 SAMN13614527 GSM4222243 WT_4705_A NA Saccharomyces cerevisiae PRJNA596180 GSE142182 Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA [RNA-Seq] ftp.sra.ebi.ac.uk/vol1/fastq/SRR107/098/SRR10720198/SRR10720198_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR107/098/SRR10720198/SRR10720198_2.fastq.gz 11 TRUE "Donczew R et al., \Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.\"", Elife, 2020 Jan 8;9""" "Log2 change values from relevant experiments for all 4900 genes were used as an input for k-means algorithm (‰Û÷KMeans‰Ûª function from Python sklearn.cluster library with default settings).;Libraries were sequenced on the Illumina HiSeq2500 platform using 25 bp paired-ends at the Fred Hutchinson Genomics Shared Resources facility.;Sequencing libraries were prepared similarly as described (Warfield et al., 2017) with several modifications;18 cycles were used for library amplification for ChEC-seq samples and 15 cycles for ChIP-samples;All libraries were sequenced on the Illumina HiSeq2500 platform using 25 bp paired-ends at the Fred Hutchinson Cancer Research Center Genomics Shared Resources facility.;All figures were generated using Matplotlib and Seaborn libraries for Python; (https://matplotlib.org; https://seaborn.pydata.org);K-means clustering was performed using ‰Û÷KMeans‰Ûª function from Python sklearn.cluster library with default settings;Two clusters were found to give the best separation using silhouette analysis (‰Û÷silhouette-score‰Ûª function from sklearn.cluster library);Resulting motifs were screened against the library of known motifs from HOMER database and the best matches were reported (Supplementary file 5);National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA " "pombe cells were mixed in an 8:1 ratio and total RNA was extracted using the RiboPure yeast kit (Ambion, Life Technologies) using the following volumes: 480 ë_l lysis buffer, 48 ë_l 10% SDS, 480 ë_l phenol:CHCl3:isoamyl alcohol (25:24:1) per S;To each 100 ë_l sample, 350 ë_l RLT lysis buffer (supplied by the Qiagen kit and supplemented with 10 ë_l 1% ë_ME per 1 ml RLT) and 250 ë_l 100% ethanol was added, mixed well, and applied to columns;Columns were washed with 500 ë_l RPE wash buffer (supplied by the Qiagen kit and supplemented 35 ë_l 1% ë_ME per 500 ë_l RPE), followed by a final 5 min spin at max speed;Newly synthesized RNA isolated via 4-thioU labeling and purification was prepared for sequencing using the Ovation SoLo or Ovation Universal RNA-seq System kits (Tecan) according to the manufacturer‰Ûªs instructions and 1 ng (SoLo) or 50 ng (Universal) input RNA" https://doi.org/10.7554/eLife.50109 TRUE
SAMN03800188 Illumina HiSeq 2500 sequencing; GSM1726241: Timecourse 1 1.5 minute labeling; Saccharomyces cerevisiae; RNA-Seq Illumina HiSeq 2500 sequencing; GSM1726241: Timecourse 1 1.5 minute labeling; Saccharomyces cerevisiae; RNA-Seq 20/11/15 Illumina HiSeq 2500 ILLUMINA PAIRED cDNA SRR2081117 SAMN03800188 GSM1726241 Timecourse 1 1.5 minute labeling NA Saccharomyces cerevisiae PRJNA288474 GSE70378 Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labelling ftp.sra.ebi.ac.uk/vol1/fastq/SRR208/007/SRR2081117/SRR2081117_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR208/007/SRR2081117/SRR2081117_2.fastq.gz 14 TRUE "Huang Y et al., \Statistical modeling of isoform splicing dynamics from RNA-seq time series data.\"", Bioinformatics, 2016 Oct 1;32(19):2965-72Barrass JD et al., \""Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling.\"", Genome Biol, 2015 Dec 17;16:282""" NA NA NA NA https://academic.oup.com/bioinformatics/article-lookup/doi/10.1093/bioinformatics/btw364 FALSE