Comprehensive RNA-Seq Analysis Pipeline

Total RNA was extracted from cell lines using RNeasy Mini Kits (Qiagen). The isolated RNA was processed and prepared using the SMARTer^® Stranded Total RNA-Seq Kit (Clontech) according to the manufacturer’s protocol. Libraries were sequenced on Illumina NextSeq using paired-end 150-bp reads. In order to remove low-quality bases, we first optimized the trimming length using mapping rate and depth of coverage (data not shown). Then, the original 2×150 paired-end reads were trimmed to 2×100 reads. We then used STAR 2.3 to align the reads to the human genome transcriptome⁶⁵ . We used RSEM 1.2.29 package to calculate the gene-level FPKM values^{66 (link)}. Differentially expressed genes were found by DESeq2 R package^{67 (link)}. Next, in order to analyze pathway enrichment, we utilized the GSEA suit (in “weighted” Enrichment statistic mode) with the inferred coverage-based ranked set^{68 (link)} (ranked by fold change).