GSM2231186: 526; Homo sapiens; Mus musculus; RNA-Seq
RNA was extracted with the Tri Reagent according to manufacturer’s instructions (Molecular Research Center Inc) Library of template molecules suitable for high throughput DNA sequencing was created following the Illumina “Truseq RNA sample preparation low throughput” protocol with some modifications. Briefly, mRNA was purified from 2 µg total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 94°C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers, then the second strand of the cDNA was synthesized using Polymerase I and RNase H. The next steps of RNA-Seq Library preparation were performed in a fully automated system using SPRIworks Fragment Library System I kit (Beckman Coulter, Fullerton, USA) with the SPRI-TE instrument (Beckman Coulter). Briefly, in this system double stranded cDNA fragments were blunted, phosphorylated and ligated to indexed adapter dimers, and fragments in the range of ~200-400 bp were size selected. The automated steps were followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C), then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation, Beverly, USA). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent, Les Ulis, France).
GEO Accession
GSM2231186