GSM2366169: Oct4 depleted; Mus musculus; RNA-Seq
Total RNA was extracted from Oct4 or Sox2 inducible ESC lines that were either untreated (wild type) or Doxycyline treated for 48hrs and 96hrs to deplete Oct4 and Sox2, respectively. Samples were DNase treated and purified using Qiagen RNeasy mini kit (according to manufacturer’s instructions). RNA quality was assessed using an Agilent 2100 Bioanalyzer. Libraries were prepared with the Truseq RNA sample preparation v2 kit. Briefly, total RNA was polyA selected using poly-T oligo-attached magnetic beads, fragmented and subject to first strand cDNA synthesis using random primers and superscript II (Invitrogen). This is followed by a second strand cDNA synthesis using polymeraseI and RNAseH. The resulting cDNA fragment were end repaired, dA-tailed at their 3’end and ligated to illumina’s multiple indexing adapters. Libraries were then PCR amplified, quantified using Qubit fluorometer and their quality assessed using an Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced on Illumina HiSeq 2000 platform. On average 20 million reads were generated per library.