RNA-Seq of Saccharomyces bayanus in log growth phase in rich media
Starting with a frozen cell pellet from our experiment, RNA extraction was performed using the MasterPure RNA Extraction Kit protocol; QC measures were taken using Nanodrop, EtBr gel, and Bioanalyzer. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 (ref. RS-122-2101/2) according to the manufacturer's protocol. 1g of total RNA were used for poly(A)-mRNA selection using streptavidin-coated magnetic beads. Synthetic spike-in transcripts (ERCC RNA Spike-In Mix 1) were added to each sample and the mixture was subsequently fragmented to approximately 300bp (range 180  500bp). cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Double-stranded DNA was purified with AMPure XP beads and was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. Purification of the final library was done with AMPure beads. Library amplification was performed by 15 cycles of PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Caliper LabChip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illuminas cBot. Libraries were then sequenced on the Illumina HiSeq 2500, Paired End, 50nts using v4 chemistry, in 1 lane, pool of 12 libraries.