Nuclear fractionation and western blot

Nuclear fractionation in mESCs was performed as described previously⁶⁰. Briefly, 10 million cells were washed by PBS and pelleted at 200 g for 2 min. 200 µl buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M Sucrose, 10% Glycerol, 0.1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail) was added to the cell pellets and incubated on ice for 8 min to remove the cytoplasm. After centrifugation at 1300 g, 4 °C, for 5 min, 100 µl Buffer N (15 mM Tris-HCl [pH 7.5], 200 mM NaCl, 60 mM KCl, 5 mM MgCl₂, 1 mM CaCl₂, 0.3% NP-40, and protease inhibitor cocktail) was added to the nuclear pellets and incubated on ice for 30 min to lyse. After centrifugation at 1700 g, 4 °C, for 5 min, the supernatant was collected and labeled as soluble fraction, and 100 µl sample loading buffer was added to the chromatin pellets for denaturing. Denatured proteins were loaded to the 4–12% gradient SDS-PAGE (GenScript). Nitrocellulose membranes (Millipore) were used for transferring after gel running. After blocking in 5% non-fat milk, the membranes were probed with the corresponding primary antibodies overnight at 4 °C, followed by incubation with a secondary antibody at room temperature for 1 h. After adding the West-Q Pico Dura ECL Solution (Gendeport), the antigen–antibody complexes were detected by the ChemiDoc Imaging system (Bio-Rad). The intensity of protein bands was measured by the Image Lab software package (Bio-Rad). The uncropped and unprocessed scans of the blots are available in source data file.