RNA-seq Protocol for Differential Gene Expression

RNA was extracted using RNAeasy kit (Bio-Rad) to perform RNA-seq as previously described^{22 (link)}. Libraries preparation, sequencing, and bioinformatics analysis of RNA sequencing were performed by Novogen (Novogen, Inc, Sacramento CA). Briefly, RNA integrity was assessed with Agilent Bioanalyzer 2100 to select RNA samples with RIN > 7.3–9.3. Two hundred fifty to 300 base pair insert cDNA libraries, non–strand-specific, were prepared with Next Ultra RNA Library Prep (New England Biolabs, Ipswich, MA) and sequenced with Illumina (San Diego, CA) HiSeq PE150 Platform ∼ 6G/sample Q30 > 90%. The reads were mapped to the human reference genome sequence using STAR v2.5 and v2.6.1, with a total mapping rate > 90% per sample. For gene expression level analysis and to calculate the fragments per kilobase of transcript per million mapped reads, HTSeq v0.6.1 was used. The differential expression analysis between two different groups was done with DESeq2 R package v2_1.6.3. The P values were adjusted using the Benjamini–Hochberg approach for controlling the false discovery rate, adjusted P < 0.05. TFCat and Cosmic databases were used to annotate the differential expressed gene. The enrichment analysis was done with cluster Profiler R package.