RNA-seq Library Preparation and Analysis

Poly-A selected libraries were prepared from total RNA at the Oxford Genomics Centre using NEBNext ultra directional RNA library prep kit for Illumina with custom 8bp indexes [70 (link)]. Libraries were multiplexed (3 samples per lane), clustered using TruSeq PE Cluster Kit v3, and paired-end sequenced (100nt) using Illumina TruSeq v3 chemistry on the Illumina HiSeq2000 platform. Samples were mapped with TopHat2 [71 (link)] on default settings with GENCODE v18 [19 (link)] as transcriptome and GRCh37 as genome reference. Exon level reads counts for all protein-coding and long non-coding transcripts present in GENCODE v18 were quantified with RNA-SeQC [72 (link)] with the “strictMode” flag set. Transcript level counts were compiled by adding up the counts for all exons. The sequenced data was required to contain at least 10M mapped and properly paired reads after applying the quality filters.