RNA-seq analysis of human transcriptome

RNA-seq was performed as previously described [9 (link)]. Total RNA was extracted using ISOGEN II reagent (Nippon Gene), according to the manufacturer's protocol. RNA quality was confirmed using the Agilent RNA 6000 Nano kit with an Agilent 2100 Bioanalyzer instrument (Agilent Technologies). RNA-seq libraries were generated using the TruSeq Stranded mRNA LT Sample Prep kit (Illumina) and sequenced with the MiSeq system (Illumina). Sequences were mapped to the human genome (GRCh38) with the Illumina Analysis Pipeline. The gene expression level was quantified by the number of uniquely mapped reads per kilobase of exon per million mapped reads (RPKM) using the Partek® Genomics Suite. Gene expression level determination and gene ontology (GO) analysis were also performed with the Partek® Genomics Suite. Significantly enriched GO functional groups were defined as having an enrichment score ≥3 (P value < 0.05). All RNA-seq data can be found online in the NCBI GEO submission (GSE157165).