Illumina HiSeq 2000 paired end sequencing; mRNA profiling of Pontin depleted ES cells and Oct4 depleted ES cells
mRNA profiling of Pontin depleted ES cells and Oct4 depleted ES cells
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.