cell type: human pluripotent stem cells, naïve shRNA: shPOU5F1
Growth protocol
Human induced pluripotent stem cells were cultured on Matrigel [BD Biosciences] in MEF conditioned medium (MEF-CM) supplemented with 8 ng/ml bFGF [Invitrogen] or maintained on irradiated feeder MEFs (iMEFs) with hPSC medium (DMEM/F12 [Gibco] with 20% Knockout Serum Replacement [Gibco], 100 mM b-mercaptoethanol, 100 mM nonessential amino acid [Gibco], 1 mM L-glutamine [Gibco], and 10 ng/ml bFGF, as previously described (Werbowetski-Ogilvie et al., 2009). Cells were routinely passaged every 5-7 days by mechanical transfer using 1 mg/ml Collagenase IV [Invitrogen]. For the derivation of naïve hPSCs, cells in primed pluripotent states were switched into hPSC medium supplemented with 1 mM MEK inhibitor PD0325901, 3 mM GSK3 inhibitor CHIR99021, and 10 ng/ml LIF (abbreviated LIF/2i) as previously reported (Buecker et al., 2010; Hanna et al., 2010; Li et al., 2009) or de novo reprogramming of somatic cells in naïve conditions. We also established naïve hPSCs using 10 ng/ml LIF and a combination of inhibitors for MEK/ERK (1 mM PD0325901), GSK3 (0.3 mM CHIR99021), LCF/SRC (1 mM WH-4-023), BRAF (0.5 mM SB590885) and ROCK (10 mM Y-27632) (5i)(Theunissen et al., 2014). Emerging colonies with a mESC-like morphology (appeared after 10-20 days of culture) were individually picked and subcultured by trypsinization or mechanical passaging onto feeder-free Matrigel or iMEFs with LIF/2i
Extracted molecule
genomic DNA
Extraction protocol
genomic DNA was extracted and purified from samples using Qiagen Dneasy Blood and Tissue Kit according to standard instructions
Label
Cy5 and Cy3
Label protocol
Standard Illumina Protocol
Hybridization protocol
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Human MethylationEpic Beadchip using standard Illumina protocol
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
