ESC culture medium.

Reagent	Amount	Final concentration
KnockOut DMEM (Gibco#10829–018)	76 mL
Fetal bovine serum embryonic stem cell qualified (ES-FBS) (Bio-Techne#S10250)	5 mL	5%
KnockOut serum replacement (KSR) (Invitrogen#10828–028)	15 mL	15%
l-glutamine (200 mM) (Gibco#25030)	1 mL	2 mM
MEM non-essentials amino acids (100 ×) (Gibco#11140–050)	1 mL	1 ×
β-Mercaptoethanol (10 mM) (Sigma#M7522)	1 mL	0.1 mM
Supplements
GSK3 inhibitor CHIR99021 (10 mM) (Stemgent#04–0004)	30 μL	3 μM
MEK inhibitor PD0325901 (10 mM) (Stemgent#04–0006)	10 μL	1 μM
LIF (107/mL) (Millipore#ESG1106)	10 μL	103/mL

Filter and store the medium (final volume 100 mL) at 4°C and use within 2 weeks. Add supplements (GSK3 inhibitor CHIR99021, MEK inhibitor PD0325901, and LIF) to the media during freshly during use of the media. ESC can be cultured in ESC media supplemented with LIF only. Prepare β-mercaptoethanol 10 mM stock from 14.3 M stock (Sigma-Aldrich M7522) by adding 6.25μL to 9 mL of sterile distilled water.