ES cells were grown in two different media, serum and 2i, defined as follow. Serum: Glasgow medium (Sigma), 15% FBS (Gibco), 2 mM L-Glutamine, 0.1 mM MEM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol, 1000 U/mL leukemia inhibitory factor (LIF, Miltenyi); 2i: 50% neurobasal medium (Gibco), 50% DMEM/F12 (Gibco), 2 mM L-Glutamine (Gibco), 0.1 mM β-mercaptoethanol, Ndiff Neuro2 supplement (Milipore), B27 serum-free supplement (Gibco), 1000 U/mL LIF, 3 μM Gsk3 inhibitor CT-99021, 1 μM MEK inhibitor PD0325901. Vitamin C (Sigma) was added at a concentration of 100 ug/mL (Blaschke et al., 2013 (link)).
When in serum, J1, Dnmt-tKO, E14, and all CRISPR-generated mutant ES cells were grown in feeder-free conditions on gelatin-coated plates. TT2, Ehmt2-KO were cultured on a monolayer of mitomycin C-treated mouse embryonic fibroblasts. ES cells were passaged with TrypLE Express Enzyme (Life Technologies, Carlsbad, CA). All 2i ES cells were grown in gelatin-coated plates and passaged every two or three days with Accutase (Life Technologies).
Trichostatin A was added for 24 hr at concentration of 25 or 50 ng/mL
Mycoplasma-free status was assessed using VenorGeM Classic mycoplasma detection kit (Minerva Biolabs).
When in serum, J1, Dnmt-tKO, E14, and all CRISPR-generated mutant ES cells were grown in feeder-free conditions on gelatin-coated plates. TT2, Ehmt2-KO were cultured on a monolayer of mitomycin C-treated mouse embryonic fibroblasts. ES cells were passaged with TrypLE Express Enzyme (Life Technologies, Carlsbad, CA). All 2i ES cells were grown in gelatin-coated plates and passaged every two or three days with Accutase (Life Technologies).
Trichostatin A was added for 24 hr at concentration of 25 or 50 ng/mL
Mycoplasma-free status was assessed using VenorGeM Classic mycoplasma detection kit (Minerva Biolabs).