Two micrograms of total RNA samples were arrayed into a 96-well plate and polyadenylated (PolyA+) messenger RNA (mRNA) was purified using the 96-well MultiMACS mRNA isolation kit on the MultiMACS 96 separator (Miltenyi Biotec, Germany) with on-column DNaseI-treatment as per the manufacturer's instructions. The eluted polyA+ mRNA was ethanol precipitated and resuspended in 10uL of DEPC treated water with 1:20 SuperaseIN (Life Technologies, USA). First-strand cDNA was synthesized from the purified polyA+ mRNA using the Superscript cDNA Synthesis kit (Life Technologies, USA) and random hexamer primers at a concentration of 5uM along with a final concentration of 1ug/uL Actinomycin D, followed by Ampure XP SPRI beads on a Biomek FX robot (Beckman-Coulter, USA). The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol by replacing the dTTP with dUTP in dNTP mix, allowing the 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 in a 96-well format using PicoGreen (Life Technologies, USA) and VICTOR3V Spectrophotometer (PerkinElmer, Inc. USA). The quality was checked on a random sampling using the High Sensitivity DNA chip Assay (Agilent). The cDNA was fragmented by Covaris E210 (Covaris, USA) sonication for 55 seconds, using a Duty cycle of 20% and Intensity of 5. Plate-based libraries were prepared following the BC Cancer Agency's Michael Smith Genome Sciences Centre (BCGSC) paired-end (PE) protocol on a Biomek FX robot (Beckman-Coulter, USA). Briefly, the cDNA was purified in 96-well format using Ampure XP SPRI beads, and was subject to end-repair and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, followed by cleanup using Ampure XP SPRI beads and 3' A-tailing by Klenow fragment (3' to 5' exo minus). After cleanup using Ampure XP SPRI beads, picogreen quantification was performed to determine the amount of Illumina PE adapters used in the next step of adapter ligation reaction. The adapter-ligated products were purified using Ampure XP SPRI beads, then PCR-amplified with Phusion DNA Polymerase (Thermo Fisher Scientific Inc. USA) using Illumina's PE primer set, with cycle conditions of 98 degree C 30sec followed by 10-15 cycles of 98 degree C 10sec, 65 degree C 30sec and 72 degree C 30sec, and then 72 degree C 5min. The PCR products were purified using Ampure XP SPRI beads, and checked with a Caliper LabChip GX for DNA samples using the High Sensitivity Assay (PerkinElmer, Inc. USA). PCR products with a desired size range were purified using a 96-channel size selection robot developed at the BCGSC, and the DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay and Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen), then diluted to 8nM. The final concentration was verified by Quant-iT dsDNA HS Assay .The libraries, 2 per 100 PE lane, were sequenced on the Illumina HiSeq 2000/2500 platform using v3 chemistry and HiSeq Control Software version 2.0.10.