GSM4633504: Circulating blood monocytes rep 2; Mus musculus; RNA-Seq
Total RNA was isolated with TRIZOL reagent according to the manufacturer's recommendation. 20ug glycogen (Ambion) was added as carrier for RNA ethanol precipitation cDNA library for RNA-Seq was generated from 100ng-400ng total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. Briefly poly-A tailed RNA molecules were pulled down with poly-T oligo attached magnetic beads. Following purification, mRNA was fragmented with divalent cations at 85C, and then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second strand synthesis was performed followed by end repair, single `A` base addition and ligation of barcode indexed adaptors to the DNA fragments. Adapter specific PCRs were performed to generate sequencing libraries. Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). Libraries were sequenced on HiSeq 2500 instrument.