Mouse feeder free E14Tg2A ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 55 µM β-mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of ESGRO (Chemicon) under feeder-free conditions. J1 ES cells and Dnmt1−/−Dnmt3a−/−Dnmt3b−/− (DNMT TKO) ES cells were maintained on gelatin-coated dishes with mitotically inactivated mouse embryonic fibroblasts. Alkaline phosphatase staining was performed with Alkaline Phosphatase Detection Kit (Chemicon). For growth curve analysis, control and knockdown ESCs were plated at 1×103 cells/cm2 and counted for 6 consecutive days. For self-renewal analysis cells were plated on 96 well plates at single cell density and the number of colonies on each plate was counted 6 days after plating.
To knockdown Tet proteins, lentiviral transduction was performed in mouse ES cells as described previously 19 (link). Short-hairpin RNA (shRNA) sequences (Supplementary Table 2) were cloned into pTY vetctor under the U6 promoter. To rescue Tet1 knockdown with Nanog, cDNA of Nanog was placed downstream of puromycin-resistant gene and foot-and-mouth disease virus 2A segment (Fig. S12), which enables multicistronic expression of transgenes in ES cells using a single promoter 21 (link).
Total RNA from mouse tissues was isolated using Trizol reagent (Invitrogen) and total RNA from cultured cells was isolated using RNeasy Mini Kit (Qiagen), and cDNA was generated with Improm-IITM Reverse Transcription System (Promega). Real-time quantitative PCR reactions were performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) using SYBR Green reagent (Invitrogen). cDNA levels of target genes were analyzed using comparative Ct methods, where Ct is the cycle threshold number and normalized to GAPDH. RT-qPCR primers are listed in Supplementary table 3.