Culturing Mouse Embryonic Stem Cells

Wild-type (WT) mESCs derived from inbred mice (Bruce 4 C57BL/6 J, male, EMD Millipore, Billerica, MA, USA) and other knock-in derivatives were cultured as described previously^{32 (link)}. C57BL/6NCr (male) mESCs^{17 (link)} were used as Sox2 STREAMING-tag knock-in cells. Briefly, all mESC lines were maintained in 2i medium (Dulbecco’s modified Eagle’s medium [DMEM, Wako, Osaka, Japan, 197-16275]; 15% fetal bovine serum [FBS, GE Healthcare, Little Chalfont, UK, SH30396.03]); 0.5 mM monothioglycerol solution [Wako, 195-15791]; 1×MEM nonessential amino acids [Wako, 139-15651]; 2 mM L-alanyl-L-glutamine solution [Wako, 016-21841]; 1,000 U/mL leukemia inhibitory factor [Wako, 195-16053]; 20 µg/mL gentamicin [Wako, 078-06061]; 3 µM CHIR99021 [Cayman Chemical, Ann Arbor, MI, USA, 13122]; and 1 µM PD0325901 [Chemscene, Monmouth Junction, NJ, USA, CS-0062]) on a 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA, G1890-100G)-coated dish at 37 °C and 5% CO₂. The cell lines used in this study are listed in Supplementary Data 1.
HeLa cells (CCL-2, ATCC) were grown in DMEM high-glucose medium (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% L-glutamine–penicillin–streptomycin solution (GPS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO₂ atmosphere.