GSM5114112: D7 CD34+CD41+ cells, SR1-, Rep2; Homo sapiens; RNA-Seq
RNAs were purified from D0 cells (isolated using Miltenyi technology), and of D7 to D14 populations (isolated by flow cell sorting) using RNeasy Plus kit (Qiagen). RNA-Seq libraries were generated from 500-300 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Two batches of RNA-seq production were generated (CNSL and VNDT series).