Total cell pellets were incubated with nuclear prep [10 mM tris-HCl (pH 7.6), 100 mM NaCl, 2 mM MgCl2, 0.3 M sucrose, 0.25% NP-40] supplemented with protein inhibitor cocktail (Roche) for 10 min at 4°C. Nuclei were collected after brief centrifugation and lysed in S300 buffer [25 mM tris-HCl (pH 7.6), 300 mM NaCl, 10% glycerol, 0.25% NP-40] supplemented with a protease inhibitor cocktail (Roche). Samples were cleared from insoluble material by centrifugation at 20,000g for 15 min at 4°C. IP was performed using anti-FLAG agarose beads (M2, Sigma-Aldrich) with overnight rocking at 4°C.Beads were then washed five times using S300 buffer. Elution was performed using 3X FLAG peptide (200 μg/ml) (APExBIO) added to the elution buffer (tris-buffered saline 1X, 50 mM NaCl) for mass spectrometry or S300 for Western blot and incubated with rocking at 4°C for 30 min. Eluted proteins were mixed 1:1 with 2X Laemmli buffer for Western blot. Input (15%) was saved before FLAG-IP and mixed 1:1 with 2X Laemmli buffer for Western blot. For mass spectrometry, eluted proteins were precipitated O/N on ice with 60 mM tris-HCl (pH 8.5) and 20% trichloroacetic acid. Tubes containing precipitated proteins were centrifuged at 20,000g for 1 hour at 4°C.The supernatant was gently discarded, and the pellet was washed twice with ice-cold acetone. Any residual acetone was carefully removed, and the protein pellet was processed for mass spectrometry. Mass spectrometry analysis was performed as described in a previous study (45).

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