title: Illumina HiSeq 2000 paired end sequencing; mRNA profiling of Pontin depleted ES cells and Oct4 depleted ES cells
design: mRNA profiling of Pontin depleted ES cells and Oct4 depleted ES cells
library_construction_protocol: Used ES cells were cultured on gelatin-coated culture dishes under feeder-free conditions.  ES cells were maintained in Dulbecco's modified Eagle medium (DMEM; Welgene), supplemented with 15% fetal bovine serum (FBS; Welgene), 0.055 mM beta-mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin, and 1,000 units/ml of LIF (Chemicon).  Pontinf/f; CreER ES cells were derived from blastocysts from intercrosses between Pontinf/f; CreER and CreER transgenic mouse. CreER transgenic mice were purchased from Jackson laboratory. Oct4-depleted ES cells were generated by treating ZHBTc4 ES cells with tetracycline for 2 days. Pontin-depleted ES cells were generated by treating Pontinf/f; CreER ES cells with hydroxyl-tamoxifen (OHT) for 3 or 4 days. We obtained total RNAs from Pontinf/f; CreER ES cells at 0, 3, or 4 days post-treatment with OHT for wt ES cells, Pontin-depleted ES cells (OHT 3D) and Pontin-depleted ES cells (OHT 4D), respectively. We also isolated RNAs from ZHBTc4 ES cells at 2 days post-treatment with tetracycline for Oct4-depleted ES cells. Libraries were prepared for multiplex sequencing using Illumina True-seq RNA sample prep kit v2, according to the manufacturer's protocol.