GSM1647131: empty-vector-RNAseq-DMSO; Homo sapiens; RNA-Seq
RNA was isolated using the RNeasy Plus Kit (Invitrogen) as directed. Plate-based libraries were prepared following the BC Cancer Agency's Michael Smith Genome Sciences Centre (BCGSC) strand specific paired-end protocol on a Biomek FX robot (Beckman-Coulter) with Ampure XP SPRI beads (Beckman-Coulter). First-strand cDNA was synthesized using the Superscript cDNA Synthesis kit (Life Technologies) with random hexamer primers and 1 μg/μL Actinomycin D. The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol, but substituting dTTP for dUTP. The cDNA was fragmented in an E210 sonicator (Covaris) for 55 seconds, using a duty cycle of 20% and intensity of 5. Purified cDNA was subjected to end-repair and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase in a single reaction, followed by 3’ A-tailing by Klenow fragment (3’ to 5’ exo minus). Products were ligated to Illumina PE adapters. The second strand was then digested using Uracil-N-Glycosylase (Life Technologies), thus achieving strand specificity. After purification, products were PCR-amplified using Phusion DNA Polymerase (Thermo Fisher Scientific) and Illumina’s PE primer set, with cycle conditions of 98°C for 30sec followed by 10-15 cycles of 98°C for 10 seconds, 65°C for 30 seconds and 72°C for 30 seconds, and then 72°C for 5 minutes. Purified PCR products were analysed using a LabChip GX (PerkinElmer). PCR products with a desired size range were purified using a 96-channel size selection robot developed at the BCGSC. The DNA quality was assessed using an Agilent DNA 1000 series II assay. DNA was quantified using a Quant-iT dsDNA HS Assay Kit (Invitrogen) and diluted to 8 nM. RNA libraries were sequenced on the Illumina HiSeq 2000/2500 platform using v3 chemistry and HiSeq Control Software version 2.0.10. RNA libraries were sequenced 2 per lane.