B27 with retinoic acid

mESCs were cultured in “Serum Free ES” (SFES) media supplemented with 2i. SFES media consists of 500 mL DMEM/F12 (Gibco #11320–033), 500 mL Neurobasal (Gibco #21103–049), 5 mL N2 Supplement (Gibco #17502–048), 10 mL B27 with retinoic acid (gibco #17504–044), 6.66 mL 7.5% BSA (Gibco #15260–037), 10 mL 100x GlutaMax (Gibco #35050–061), and 10 mL 100x Pen/Strep. To make “2i SFES”, 1 nM PD03259010 (Selleckchem #S1036), 3 nM CHIR99021 (Selleckchem #S2924) and 1000 units/mL LIF (ESGRO #ESG1106) were added to 45 mL SFES. Prior to use, 1-thioglycerol (MTG; Sigma M6145) was diluted 1.26% in SFES and added 1:1000 to 2i SFES media. To passage, mESCs were treated with 1 mL of accutase in a 6 well plate (Corning #353046) for 5 minutes at room temperature (RT). After incubation, cells were mixed by pipette and moved to a 15 mL conical tube, supplemented with 10 mL SFES and spun at 300xg for 3 minutes. Then, media was removed and cells were counted using the Countess II Cell Counter (ThermoFisher) according to the manufacturer’s instructions. Cells were then plated in 6 well plates that had gelatinized with 1% gelatin for 30 minutes at 37 C at 5 × 10^5 cells per well in 2 mL of 2i SFES. Media was changed every day and cells were split every other day. Cells and culturing reagents were gifted by Dr. Abigail Buchwalter.