Bulk RNA-Seq Protocol for Differential Gene Expression

The extraction and purification of total RNA was performed using the RNAeasy purification Kit (QIAGEN) combined with RNaseFree DNase (QIAGEN) treatment according to the manufacturer’s protocol. RNA concentration and quality was determined using the NanoDrop 2000 spectrophotometer before downstream processing. Quality of the purified RNA was tested on an Agilent 2200 Tapestation using RNA screentape. Libraries were prepared using the TruSeq stranded total RNA with RiboZero kit (Illumina). Quality and quantity of the cDNA libraries were assessed on an Agilent 2200 Tapestation (D1000 screentape). Libraries were run on an Illumina NextSeq500 using the High Output 75 cycles kit (2 × 36 cycles, paired-end reads, single index). Quality checks on the raw RNA-Seq data files were done using FastQC [52 (link)]. Alignment of the RNA-Seq paired-end reads was to the GRCm38 [53 (link)] version of the mouse genome and annotation using HiSat2 [54 (link)] and TopHat [55 (link)]. Gene expression levels were quantified using the feature Counts tool [56 (link)] available on Galaxy.org DESeq2 [57 (link)] was used to generate a list of differentially expressed protein-coding genes between. Calculations were performed with R version 3.3.1 in R-Studio (version 0.99.903). RNA-Seq data is available as GEO accession number GSE190668.