RNA-seq Data Analysis Protocol

RNA was isolated (Machery Nagel NucleoSpin RNA Kit, Dueren, Germany) and quality was assessed with NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Bioanalyser measurements (Agilent, Santa Clara, CA, USA). The RNA sequencing data quality was assessed using FastQC (V0.11.4, Babraham Institute, Cambridge, UK) [53 ] before aligning reads with STAR (V2.5.4b) [54 (link)] against the Ensembl H. sapiens genome V91. The alignment quality was analyzed using samtools (V1.1) [55 (link)]. For all genes, normalized read counts were obtained using GenomicAlignments (V1.14.2) and DESeq2 (V1.18.1) [56 (link)]. Transcripts covered with <50 reads were excluded from subsequent analyses. The significance thresholds were set to |log2 fold-change| ≥ 1 and BH-adjusted p ≤ 0.01. To minimize sample variations, surrogate variable analysis (sva, V3.26.0) was used [57 (link)]. nRPKMs (normalized reads per kilobase per million total reads) were calculated from raw counts from DESeq2 [58 (link)]. Using gene sets provided by Andersson et al. and Stam et al. [59 (link),60 (link)], gene set enrichments were determined with GSEA (V3.0) [61 (link),62 (link)]. RNA-seq data were deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE128342.