Bard1

Cells were harvested and then lysed with RIPA lysis buffer containing 1.0 mM protease inhibitor cocktail, 1.0 nM DTT and PMSF for 30 min at 4 °C. Then, the protein supernatant was collected using a microcentrifuge at 13,000 rpm for 15 min at 4 °C. Appropriate protein (50 μg) of samples were subjected to 10–12% SDS-PAGE gel electrophoresis and electroblotted into PVDF membranes (Millipore, Darmstadt, Germany) as previously described^{54 (link)}. The membranes were blocked with skimmed milk solution (5%) and incubated with the following primary antibodies: GAPDH (1:3000; Origene, USA), β-Actin (1:3000; Abclonal, USA), Osteocalcin (1:1000; Santa Cruz Biotechnology, USA), RUNX2 (1:1000; Proteintech, China), SP7/Osterix (1:1000; Abcam, USA), BARD1 (1:500; Santa Cruz Biotechnology, USA), FBXO25 (1:300; Santa Cruz Biotechnology, USA), CUL3 (1:500; Santa Cruz Biotechnology, USA), H2A (1:1000; Absin, China), H2B (1:1000; Abcam, USA), H3 (1:1000; CST, USA), H2AK119ub (1:1000; CST, USA), H2BK120ub (1:1000; CST, USA), and H3K4me3 (1:2000; CST, USA). The results were visualized using a MiniChemi 610 System Instrument (Beijing, China).

HeLa, U2OS and HEK293Tcells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) were cultured in DMEM (Invitrogen) supplemented with 10% FBS (HyClone) as recommended. H2AX^+/+ and H2AX^−/− MEFs were a gift from Andre Nussenzweig, and MDC1 proficient and deficient MEFs were a gift from Zhenkun Lou. GM00200 (ATM + / + ) and GM09607 (ATM −/−_) were obtained from Coriel. Cells were grown at 37 °C and in a humidified chamber with 5% CO_2.Drugs and Antibodies: G9a inhibitor UNC0638 (Sigma-Aldrich), ATM kinase inhibitor KU-55933 (Astra-Zeneca/Kudos Pharmaceuticals (Cambridge, United Kingdom), 0.5 μM), Neocarzinostatin (NCS, Sigma Aldrich, 200 ng/ml,), and ATR inhibitor VE-821 (Cayman chemicals, 3 μM). Antibodies used were: G9a (Cell Signaling Technology), GLP1 (Bethyl Laboratories), phosphorylated (Ser139) H2AX (EMD Millipore), 53BP1 (Bethyl Laboratories), BRCA1 (Gift from Bing Xa), BARD1 (Santa Cruz Biotechnology), anti-PAR (Travigen), SUMO (Santa Cruz Biotechnology), H3K9me2 (EMD millipore), Flag (Sigma Aldrich), SPOC1 (Sigma Aldrich), K63-ubiquitin (EMD Millipore), GFP (Abcam) and GAPDH (Abcam), For immunofluorescence experiments, the secondary antibodies used were: anti-mouse (115-035-068, 1:20,000) and anti-rabbit (711-035-152, 1:20,000) from Jackson ImmunoResearch Labs.

The following antibodies were used: RNAPII CTD S2 (Bethyl cat# A300-654A, 1:10,000); RNAPII CTD S5 (Bethyl cat# A300-655A, 1:10,000); RNAPII (Santa Cruz cat# sc-899, 1:1000); RNAPII CTD S7 (Millipore cat# 041570, 1:10,000) RNAPII CTD Thr4 (Active Motif cat# 61361, 1:1000) cleaved PARP (Cell Signaling cat# 9541, 1:1000); β-Actin (Cell Signaling cat# 4967, 1:4000); CDK12 (Cell Signaling cat# 11973 s, 1:1000); CDK13 (Bethyl cat# A301-458A, 1:1000); CDK13 antibody (1:1000) used for WB in Supplementary Fig. 2e was kindly provided by Dr. Arno Greenleaf; γ-H2AX (Cell Signaling cat# 9718, 1:200), RAD51 (GeneText cat# GTX70230, 1:1000), BRCA1 (Cell signaling cat# 9010S, 1:500), BARD1 (Santa Cruz Biotechnology cat# sc11438, 1:500), Alexa-488 (Molecular Probes cat#A11008, 1:300).