FFPE RNA-Seq Library Preparation and Analysis

RNA was extracted from FFPE tissue using the automated Maxwell system (Promega, Madison, USA). RNASeq libraries were prepared using the Illumina TruSeq Stranded Total RNA library prep, with Ribozero Gold, starting from 500 ng of DNAse I treated total RNA, following the manufacturer’s protocol, with the exception that 14 cycles of PCR were performed to amplify the libraries, to keep the duplication rate lower than with the recommended 15 cycles. The amplified libraries were purified using AMPure beads, quantified by Qubit and QPCR, and visualized in an Agilent Bioanalyzer. The libraries were pooled equimolarly, and loaded at 8 pm, on a high output HiSeq 2500 flow cell, as paired 50 nucleotide reads. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were aligned to the human genomes (build hg19/GRCh37) using the splice-aware STAR aligner^{29 (link)}. The featureCounts program^{30 (link)} was utilized to generate counts for each gene based on how many aligned reads overlap its exons. These counts were then normalized and used to test for differential expression using negative binomial generalized linear models implemented by the DESeq2 R package^{31 (link)}.