Naïve murine embryonic stem cells (mESC) were derived freshly (mixed 129/B6, XY), or obtained from (Hackett et al, 2018 (link)), and were routinely cultured on gelatin‐coated plates in t2i/L media: NDiff (N2B27) (Takara #y40002) supplemented with titrated 2i (0.2 μM PD0325901 and 3 μM CHIR 99021), 1,000 U/ml leukaemia inhibitory factor (LIF), 1% FBS, 1% penicillin streptavidin and maintained in humidified atmosphere at 37°C and 5% CO2. Cells were passaged every 2–3 days by dissociation with TrypLE and medium was changed daily. Mycoplasma contamination checks were performed routinely by ultrasensitive qPCR assay (Eurofins).
For X‐chromosome reactivation experiments, TX1072 cells (mixed Cast/B6, XX) were a gift from Edith Heard, and were cultured as described before in 2i+Lif media: DMEM (Sigma), 15% FBS (Gibco), 1% penicillin streptavidin, 0.1 mM β‐mercaptoethanol, 1,000 U/ml (LIF), CHIR99021 (3 μM) and PD0325901 (1 μM).
For X‐chromosome reactivation experiments, TX1072 cells (mixed Cast/B6, XX) were a gift from Edith Heard, and were cultured as described before in 2i+Lif media: DMEM (Sigma), 15% FBS (Gibco), 1% penicillin streptavidin, 0.1 mM β‐mercaptoethanol, 1,000 U/ml (LIF), CHIR99021 (3 μM) and PD0325901 (1 μM).