SLX-13090.A021.HKMKJBBXX.s_3.r_1.fq
Total RNA was isolated from cells using the RNeasy Kit (Qiagen) according to the manufacturer's protocol. An additional DNase1 digestion step was performed to ensure that the samples were not contaminated with genomic DNA. One microgram of total RNA, after validation by an Agilent Bioanalyser 2100 system to have an appropriate RIN between 7.6-10, was used for library construction using Illumina's TruSeq Stranded mRNA Library Prep Kit (Illumina, CA, U.S.A) at the Institute of Metabolic Science Genomics and Transcriptomics Core Facility (Cambridge, UK).  Messenger RNA was enriched by poly-T oligo attached magnetic beads before reverse transcription.   Addition of a single A nucleotide (adenylation) after the synthesis of the double-stranded cDNA stopped the ligation of DNA fragments during the adapter ligation reaction.  Unique barcodes were added to individual samples allowing multiplexed sequencing.  DNA fragments with successful adapters ligated were enriched with a limited amplification.  The second strand cDNA, with the incorporation of dUTP, was quenched during amplification to allow strand-specific sequencing. Indexed libraries were purified, normalised, pooled and sequenced on the Illumina HiSeq 4000 platform at single read length of 50 base pairs at the Genomics Core Facility, Cancer Research UK Cambridge Institute (Cambridge, UK).
Combi-4