Co-immunoprecipitation

For co-immunoprecipitation assays cell media were supplemented with 5 mM Sodium Butyrate 24 h prior collection. RIP was performed using 10⁷ cells/antibody condition. Cell nuclei were isolated using the Nuclei Isolation Buffer (NIB: 0.256 M Sucrose, 8 mM Tris-HCl pH 7.5, 4 mM MgCl₂, 1% Triton X-100, cOmplete™ Mini Protease Inhibitor Cocktail [Roche], 1 mM PMSF) supplemented with 20 mM Sodium Butyrate for 30 min on ice followed by dounce homogenization. After centrifugation at 2500 × g for 15 min at 4 °C, the supernatants were discarded and the nuclei pellet was washed with PBS supplemented with cOmplete™ Mini Protease Inhibitor Cocktail (Roche), PMSF (1 mM), and cleared by centrifugation at 2500 × g for 15 min at 4 °C. Nuclei enriched pellet was further lysed in RIPA buffer (150 mM KCl, 25 mM Tris-HCl pH 7.4, 5 mM EDTA, 0.5 M DTT, 0.5% NP40, cOmplete™ Mini Protease Inhibitor Cocktail [Roche], PMSF) supplemented with Benzonase® nuclease (Sigma Aldrich, final concentration 1 U/ml), 20 mM Sodium Butyrate, and 1 mM MgCl₂ on ice for 40 min. Nuclei lysates were cleared by centrifugation at 13,000 × g for 10 min at 4 °C. Supernatants were pre-cleared by incubation with Protein G-sepharose beads (GE Healthcare) for 2 h at 4 °C. To couple the antibodies to the Dynabeads™ Protein G (Invitrogen), indicated antibodies (Anti-HDAg antibody-positive human serum, Anti-BAZ2B [Life technologies, Cat#: 730006, RRID:AB_2532814, final concentration 30 µg/ml], Anti-SNF2L [Abcam, Cat#: ab37003, RRID:AB_945532, final concentration 10 µg/ml], Anti-SNF2H [Abcam, Cat#: ab3749, RRID:AB_2191856, 10 µg/ml], or nonspecific control Rabbit antibody IgG [Abcam, Cat#: ab171870, RRID:AB_2687657, 20 µg/ml]), were added to 50 µl of beads resuspended in 0.5 ml of Antibody Coupling Buffer (ACB: 3% BSA, cOmplete™ Mini Protease Inhibitor Cocktail [Roche], 1 mM PMSF) overnight at 4 °C, one day prior to the RIP assay. To remove the uncoupled antibodies, beads were washed thrice with ACB and equilibrated in 100 µl of ACB. Pre-cleared nuclear lysates were next incubated overnight at 4 °C with the beads coupled to the indicated antibodies. After thorough washing with RIPA buffer, beads were resuspended in Laemmli buffer (2×) and incubated at 95 °C for 10 min. Immunoprecipitated samples were resolved using 4–20% precast SDS polyacrylamide gels. Horseradish peroxidase-linked secondary antibodies (Veriblot [Abcam, Cat#: 131366, 1:1000 dilution]) were detected by chemiluminescence. For co-immunoprecipitation assays involving the GFP-TRAP®_M beads (Chromotek), the nuclei lysates were obtained as described above and incubated with 25 µl of GFP-TRAP®_M beads overnight at 4 °C. After thorough washing with RIPA buffer, beads were resuspended in Laemmli (2×) and incubated at 95 °C for 10 min prior to electrophoresis.