Truseq pe v3 flowcells

Using 500 ng of total-RNA from each of the samples, RNA-Seq libraries were constructed by depletion of rRNA, followed by synthesis of directional, paired-end, and indexed RNA-Seq libraries. The rRNA-depleted RNA was purified using the RNA Clean & Concentrator-5 columns (Zymo Research). Speed-vac was used to reduce the remaining sample volume to 9.5 μL, followed by synthesis of directional, paired-end, and indexed RNA-Seq libraries using the ScriptSeq Kit (Epicentre). Briefly, rRNA-depleted RNA was chemically fragmented, and cDNA was synthesized from a tagged random hexamer. The cDNA was terminal tagged using a 3′-end blocked and tagged oligo, followed by MiniElute (Qiagen) purification. The di-tagged cDNA was then used as template in a 10-cycle PCR reaction. Libraries were purified by agarose gel electrophoresis selecting elements of 200–600 bp in size. The RNA-Seq libraries were denaturated and diluted to 10 pM with pre-chilled Hybridization buffer (Illumina) and loaded into TruSeq PE v3 flowcells (Illumina) on an Illumina cBot, followed by indexed paired-end sequencing (101 + 7 + 101 bp) on a Illumina HiSeq 2000 using TruSeq SBS Kit v3 chemistry.