mESC were cultured as previously described (Brosh et al. 2023 (link)). Specifically, C57BL/6J × CAST/EiJ (B6xCAST) mESCs were cultured on plates coated with 0.1% gelatin (EMD Millipore ES006-B) in 80/20 medium comprising 80% 2i medium and 20% mESC medium. 2i medium contained a 1:1 mixture of Advanced DMEM/F12 (ThermoFisher 12634010) and Neurobasal-A (ThermoFisher 10888022) supplemented with 1% N2 Supplement (ThermoFisher 17502048), 2% B27 Supplement (ThermoFisher 17504044), 1% GlutaMAX (ThermoFisher 35050061), 1% Pen-Strep (ThermoFisher 15140122), 0.1 mM 2-mercaptoethanol (Sigma M3148), 1,250 U/mL LIF (ESGRO ESG1107l), 3 μM CHIR99021 (R&D Systems 4423), and 1 μM PD0325901 (Sigma PZ0162). mESC medium contained knockout DMEM (ThermoFisher 10829018) supplemented with 15% FBS (BenchMark 100–106), 0.1 mM 2-mercaptoethanol, 1% GlutaMAX, 1% MEM nonessential amino acids (ThermoFisher 11140050), 1% nucleosides (EMD Millipore ES008-D), 1% PenStrep, and 1,250 U/mL LIF. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and passaged twice per week on average.