4.11. RNA Sequencing of Fresh Frozen Tissues
In total, 34 RNA samples from 29 patients were sequenced in 2 batches. All samples showed RIN > 3. Ribosomal depletion and RNA libraries were constructed with Ribo-Zero rRNA-depleted stranded (Illumina, San Diego, CA, USA) and the NEBNext adaptors, starting with 250 ng of total RNA. Libraries were sequenced as 125 bp paired-end reads on Illumina HiSeq 4000 (Genome, Quebec, QC, Canada). Reads were trimmed using Trimmomatic (v0.35) (Bolger, AM 2014 2114), removing the adaptor, the first four bases from the start of each read, and low-quality bases at the end of each read using a 4 bp sliding window to trim where average window quality fell under 30. Trimmed reads < 30 bp were discarded. Clean reads were then aligned to the reference genome hg19 (GRCh37) using STAR v2.3.0 (Dobin, A. 2013; 15). Raw gene counts were obtained using featureCount (Liao, Y. 2014-923), on the UCSC hg19 annotation. Differential expression analysis was performed using DESeq2, and the batch information was included in all designs for outcome or response group-based comparison. Genes were considered significantly differentially expressed if they met the following criteria: average expression across samples ≥ 50, |log2 Fold Change| ≥ 1, and false discovery rate (FDR) ≤ 0.05. CMS classification was performed using the CMSclassifier R package [