Computational protocol: Histone methyltransferase SETD2 coordinates FACT recruitment with nucleosome dynamics during transcription

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Protocol publication

[…] We analyzed previously reported poly(A)+ RNA-seq data from human mesenchymal stem cells transfected with control and targeting siRNAs directed against SETD2 (). High-throughput sequencing reads were aligned to the reference human Genome hg19 using TopHat (), removing reads mapping to multiple locations. Our analysis was restricted to transcriptionally active genes defined as the genes with at least 50 reads/kb in both samples. Gene annotations were obtained from UCSC knownGene table (). To identify genes with intragenic transcription initiation, we compared read counts for expressed exons (minimum of 5 reads/100 bp required) in depleted and control cells. First, we selected all transcripts with significantly higher read counts for at least one exon in SETD2-depleted cells. Second, we discarded transcripts with significantly higher read counts for the first annotated and expressed exon (likely corresponding to upregulated full-length mRNAs), Third, we defined the new cryptic TSS as the first significantly higher exon and filtered out all transcripts that showed significantly higher read counts for <60% of the downstream exons (possibly corresponding to isolated alternative splicing events). Significantly higher exons were defined as those having exonic read counts (normalized reads per million) fold change higher than 1.2 between SETD2-depleted and control cells, and Fisher’s proportion test P < 0.05 (alternative hypothesis: read count proportions relative to the total library size in siSETD2 greater than siControl). To withdraw restrictions by previous gene annotation, we performed transcriptome assembly using Cufflinks (). […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Saccharomyces cerevisiae, Homo sapiens