TS cells were cultured in RPMI 1640 supplemented with 20% fetal bovine serum, 1 mM sodium pyruvate (Invitrogen), 1X penicillin/streptomycin, 55nM β-mercaptoethanol (Invitrogen), 25 ng/mL fibroblast growth factor 4(FGF4; Sigma), and 1 μg/mL heparin (Sigma) with 70% of medium being conditioned for 72hrs on inactivated mouse embryonic fibroblasts. ES cells were cultured in DMEM with high glucose supplemented with 15% FBS, GlutaMAX, ß-mercaptoethanol, sodium pyruvate, non-essential amino acids, and 1000U/ml of LIF (Leukaemia inhibitory factor). MEFs were cultured in DMEM supplemented with 10% FBS and non-essential amino acids. Epiblast stem cells were cultured in CDM consisted of Iscove’s modified Dulbecco’s Medium plus Ham’s F12 medium at a 1:1 ratio, both supplemented with Glutamax, bovine serum albumin at a final concentration of 5 mg/ml, 1X chemically defined lipid concentrate, transferrin, insulin at 7 ug/ml.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified using the MirVana RNA extraction kit (Ambion). Total RNA was quantified using the Ribogreen reagent (Lifetech, Inc.), and quality-controlled on a Bioanalyzer (Agilent). Two hundred nanograms of input total RNA was amplified and labeled using the TotalPrep kit (Ambion). The labeled product was then hybridized to Illumina HT12 arrays and scanned on a BeadArray Reader (Illumina, Inc.) according to the manufacturer’s instructions.
Label
Biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Data processing
The data were filtered for detection pValue > 0.01 and normalised using Robust Spline Normalisation with lumi in R
