RNA-seq Protocol for Transcriptome Analysis

RNA sequencing was performed by the Nucleomics Core Facility (VIB, Leuven, Belgium). RNA was isolated using an RNeasy kit (Qiagen). From extracted RNA, libraries were made using the Illumina TruSeq Stranded mRNA Library protocol. These libraries were sequenced on an Illumina NextSeq 500 paired-end 75 bp and yield an average of 90.2 million reads per sample (range 75.6–106.9). To estimate the expression of the transcript of every sample, reads were counted using Salmon (v0.8.1)^{68 (link)} against the ensembl transcript for the human reference genome hg38. Gene expression from the protein coding transcripts was then estimated using the tximport function the R-package tximport (v1.6.0)^{69 (link)}.