Splicing factor BUD31 promotes ovarian cancer progression through sustaining the expression of anti-apoptotic BCL2L12

Custom code and analysis pipelines used in the paper are shown in markdown files.

• AS_pipeline.md This file includes intructions for using rMATS, MISO, and Spliceseq to analyze individual alternative splicing (AS) events. Besides, SpliceR is a classcical software for NMD (nonsense-mediated mRNA decay) annotation and genomic elements length calculation.
• RNA-seq_pipeline.md The adaptor sequence and low-quality reads were removed with TrimGalore (version 0.6.1). Clean reads were aligned to the hg38 genome with HISAT2 (version 2.2.0) and sorted with samtools (version 1.9). Gene expression quantification was performed by counting reads over genes from the same annotation as alignment using featureCounts (version 2.0.0).Mapped reads were visualized with the Integrative Genomics Viewer (IGV). Transcripts were reconstructed with StringTie (version 1.3.0). Differential expression was analyzed with edgeR (version 3.36.0.) or DESeq2 (version 1.32.0).
• SpyCLIP_pipeline.md Crosslinking sites were identified using the previously described iCLIP analysis pipeline. The adaptor sequence and low-quality reads were removed with TrimGalore (version 0.6.1), and the quality of the clean reads was checked with FastQC (version 0.11.9). rRNA sequences were removed with bowtie (version 1.2.3). The remaining reads were mapped to the human genome (hg38) using the STAR software (version 2.7.1a). PCR duplicates of uniquely mapped reads were removed using Picard (version 2.25.5) with MarkDuplicates. The remaining reads were considered usable reads for identifying crosslinking sites. Mapped reads were visualized in IGV. Two technical replicates of the SpyCLIP samples were merged for PURECLIP (version 1.2.0) analysis with -ld -nt 16 -dm 80. Individual crosslink sites (< 80 nt) were merged as raw binding regions. Binding regions that acquired more than three crosslink sites were used for further analysis as previously suggested, and these regions were annotated using HOMER (version 4.11) into exon, intron, promoter, intergenic, 5’UTR, 3 ’UTR, etc. The 80-nt regions around the center of each binding region were extracted and used to identify the de novo BUD31 binding motif using HOMER’s findMotifs program (-len 6,8,10,12 -S 10 -rna -p 4). Motifs were matched to the genome position with scanMotifGenomeWide.pl function belonging to HOMER and visualized with Deeptools (version 3.1.3).