Truseq stranded total rna library prep

RNA integrity number (RIN) was established for each sample by using the Agilent 2200 TapeStation system (Agilent Technologies, 2503). Control and WD samples (n = 5 for each condition) had an average RIN value of 6.27 (range 5.2–7). cDNA libraries were prepared using the TruSeq® Stranded Total RNA Library Prep (Illumina, 15031048) following the manufacturer’s instructions. The ribosomal RNA was depleted from the total RNA sample using rRNA removal beads. Following purification, the depleted samples were broken into small fragments (75 bp) that were used for first strand cDNA synthesis and a subsequent second strand cDNA synthesis. The cDNA fragments then went through a single adenine addition and ligation of adapter indices. Library amplifications were performed by PCR to generate the final cDNA libraries. Sample with average cDNA library size close to 260 bp (following the manufacturer’s protocol) and higher final library concentration was used for RNAseq. Approximately 200 ng individual libraries were sequenced using the NextSeq500 High Output Version 2.5, 2 × 75 bp kit (Illumina, 15057931) following the manufacturer’s manual. The samples generated averagely 22.2 million mapped reads per sample (range 17.7–37 million). Data was processed using the Real-Time Analysis (RTA) software (version 2.4.6).

Libraries were prepared using the Illumina TruSeq Stranded Total RNA library prep, with Ribozero Gold, starting from 500 ng of total RNA and loaded on high output HiSeq 2500 flow cells. RNA alignments were performed using STAR and reads mapping to exons counted using featureCounts. Counts were normalized in R using the DESeq2 package.
Detailed protocols can be found in the supplementary information file.

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)}.