Co-immunoprecipitation of Tet1 in ESCs

Co-immunoprecipitation experiments were performed as described before (29 (link),30 (link)). Briefly, ESCs were washed and collected in ice-cold PBS supplemented with Halt PIC (Thermo 78430). Nuclear extracts were prepared and treated with benzonase nuclease (Sigma, E1014), and incubated with 4 μg of antibody (anti-Tet1 (GeneTex, GTX125888), Rabbit-IgG (CST, 3900) crosslinked to Protein G-conjugated magnetic beads (Dynabeads protein G, Invitrogen) overnight at 4°C. IgG was used as control. Immunocomplexes were washed with buffer containing 20 mM HEPES, pH 7.6, 10% glycerol, 100 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA. The proteins were eluted in 2× Laemlli buffer at 95°C and analyzed by western blot as described above.