Generation of Tet1/2/3 Triple Knockout mESCs

mESCs (E14) were cultured on MEFs in Knock-out Dulbecco’s Modified Eagle’s Medium (Gibco), supplemented with 15% fetal bovine serum (Omega), 0.5% penicillin-streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM 2-mercaptoetanol (Sigma), and 103 U/mL of leukemia inhibitory factor (Millipore). Tet1/2/3 triple knock-out mESCs were generated by the CRISPR/Cas9 technology as described previously^{44 (link)} with slight modifications. Tet1/2/3 sgRNAs were cloned into PX458 (Addgene 48138). Three sgRNAs were transfected simultaneously into mESCs using the iMfectin DNA transfection reagent (Gendepot). mESCs transfected with the vector PX458 without sgRNA were used as control. GFP positive cells were sorted into 96-well plates by flow cytometry and individual colonies were genotyped after 7-day culture.