RNA sequencing (RNA-seq) and data analysis

RNA extraction and RNA-seq library preparation upon GFP and NCBP3 depletion were performed as described (44). Total RNAseq of siNCBP3 depletion samples are first reported here, but were collected as part of the same experiment described in (44,45). Reads from siNCBP3 samples were processed in parallel with the siEGFP control and relevant depletions from GEO:{"type":"entrez-geo","attrs":{"text":"GSE99059","term_id":"99059"}}GSE99059 were processed in parallel as described in (46). In brief, raw reads were quality filtered and trimmed as described (29), using Trimmomatic (v 0.32) and settings (PE ILLUMINACLIP:/com/extra/Trimmomatic/0.32/adapters/TruSeq3-PE-2.fa:2:30:10 HEADCROP:12 LEADING:22 SLIDINGWINDOW:4:22 MINLEN:25). Cleaned reads were then mapped to GRCh38 with HISAT2 (v 2.1.0) (47) using default settings and the genome index 'H. sapiens, UCSC hg38 and Refseq gene annotations’ provided at the HISAT2 download page (ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/hg38_tran.tar.gz). Only proper pairs with both reads mapping to the genome were used for further analysis. Exon read counts were collected using the featureCounts tools from the subread software suite (v 2.0.0) (48) with settings ‘-p -C -s 2 -t exon’ for the default GRCh38 annotations for featureCounts. Differentially expressed transcripts were obtained from raw read counts using R package DESeq2 (v 1.20.0) at a false discovery rate (FDR) cutoff of 0.1. Exon counts for differentially up- and down-regulated transcripts were compared using custom scripts in R.