RNA-seq protocol for differential gene expression analysis

Total RNA was extracted using GenElute mammalian total RNA miniprep kit (Sigma) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase (Sigma). RNA integrity was assessed by NanoDrop, Qubit assay, and/or using an Agilent 2100 bioanalyzer per each manufacturer’s specifications. Sample amounts were normalized, and 1,000 ng was used for library preparation using the NEB Ultra RNA Library Preparation kit per the manufacturer’s instructions. Library quality control (QC) and quantitation were performed on all individual libraries by the Qubit assay and by using the Agilent 2100 bioanalyzer. Libraries were normalized and pooled via Qubit measurement. The final pool was quantitated via quantitative PCR (qPCR). Sequencing was performed on the Illumina HiSeq2500 rapid-run mode on one flow cell (two lanes) per the system manufacturer. Raw RNA-seq data were processed, normalized, and mapped to the human reference genome (hg19) using CLC Genomics Workbench 8 (Qiagen). Differentially expressed genes were identified using DESeq2 (38 (link)) with the indicated significance cutoffs. Hierarchical clustering was performed using Cluster 3.0/Java Treeview and heat maps were generated using MeViewer software (17 (link)).
Pathway analysis was performed using MetaCore by GeneGo, with a statistical cutoff of P < 0.001 applied for pathway enrichment.