title: RNA-seq analysis of RA (UT, 1h, Rep1) regulated genes in mouse embryonic stem cell
design: RNA-Seq library was prepared from two biological replicates by using TruSeq RNA Sample Preparation Kit (Illumina) according to manufacturer’s protocol. Libararies were prepared from 2.5?g total RNA. In the first step poly-A tailed RNA molecules (mRNA) were purified with poly-T oligo-attached magnetic beads. Following the purification mRNA is fragmented using divalent cations at 85ºC degree, then first strand cDNA was generated using random primers and SuperScript II reverse transcriptase (Invitrogen, Life Technologies). This is followed by second strand cDNA synthesis, then double stranded cDNA fragments went through an end repair process, the addition of a single ‘A’ base and then barcode indexed adapter ligation. Adapter-ligated products were enriched with adapter specific PCR to create cDNA library. Agarose gel electrophoresis was performed on E-Gel EX 2% agarose gel (Invitrogen, Life Technologies) and the library was purified from the gel using QIAquick Gel Extraction Kit (Qiagen). Fragment size and molar concentration were checked on Agilent BioAnalyzer using DNA1000 chip (Agilent Technologies). Libraries were sequenced on HiSeq 2000 with paired-end protocol based on manufacturer’s instruction.