RNA-seq Analysis of Human Transcriptome

TRIzol reagent (Invitrogen, CA, USA) was used for RNA extraction, and cDNA libraries were prepared based on the TruSeq^® Stranded Total RNA Sample Preparation Guide (Illumina, Part # 15031048, San Diego, CA, USA). Equal concentrations of each library were sequenced using a NextSeq 500 (Illumina) platform to create pair-end 75-bp reads. Quality assessment and trimming of the generated sequences were done by the RNA-seq alignment tool from BaseSpace (Illumina), followed by alignment to the human reference genome (hg38) with STAR 2.5.2b [21 (link)]. The expression levels of genes in each sample and the corresponding fold changes were estimated by DESeq2 1.14.1 (Partek Genomics Suite, St. Louis, MO, USA) [22 (link)] with GENCODE V19 (V25) annotation [23 (link)]. Relative expression of each gene is represented by CPM (Counts Per kilobase Million). Partek Genomics Suite and statistical package were used for the statistical analysis, hierarchical clustering differential expression analysis, and Gene ontology (GO) enrichment. Canonical pathway analysis was performed by using the Ingenuity Pathway Analysis (IPA).