E210 sonication

Total RNA samples were analyzed using an Agilent Bioanalyzer RNA nanochip and 2ug arrayed into a 96-well plate. PolyA+ RNA was purified using the 96-well MultiMACS mRNA isolation kit (Miltenyi Biotec, Germany). The eluted PolyA+ RNA was ethanol precipitated and re-suspended in 10µL of DEPC treated water with 1:20 SuperaseIN (Life Technologies, USA). First-stranded cDNA was synthesized from the purified polyA + RNA using the Superscript cDNA Synthesis kit (Life Technologies, USA). The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol by replacing the dTTP with dUTP in dNTP mix, allowing second strand to be digested using UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction and thus achieving strand specificity. The cDNA was quantified using PicoGreen (Life Technologies, USA) and fragmented by Covaris E210 sonication. The paired-end sequencing library was prepared following the BC Cancer Agency Genome Sciences Centre strand-specific, plate-based and paired-end library construction protocol on a Biomek FX robot (Beckman-Coulter, USA). The 75 base PE libraries were sequenced on Illumina HiSeq2000 instruments. Analysis of mRNA expression was determined as previously described.(25 (link))