Epidyne nucleosome remodeling assay substrate st601 gatc1

SMARCA4 (BRG1) levels of the HA-purified mSWI/SNF complex purifications were normalized via BCA protein quantification and Silver Stain analyses. Purified mSWI/SNF complexes were diluted for final reaction concentration of 10 ng/μL in REAA buffer (20 mM HEPES, pH 8.0, 50 mM KCl, 5 mM MgCl₂) containing 0.1 mg/mL BSA, 1 μM DTT, 20 nM nucleosomes (EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, EpiCypher). The REAA mixture was incubated at 30 or 37°C for 10 min, and reaction was initiated using 1-2 mM ATP (Ultrapure ATP, Promega) and 0.005 U/μL DpnII Restriction Enzyme (New England Biolabs). The REAA reaction mixture was quenched with 20-24 mM EDTA and placed on ice. Proteinase K (Ambion) was added at (100 μg/mL)_for 30-60 min, followed by either AMPure bead DNA purification and D1000 HS DNA ScreenTape Analysis (Agilent) or mixing with GelPilot Loading Dye (QIAGEN) and loading onto 8% TBE gel (Novex 8% TBE Gels, Thermo Fisher). TBE gels were stained with either SYBR-Safe (Invitrogen) or Syto-60 Red Fluorescent Nucleic Acid Stain (Invitrogen), followed by imaging with UV light on an Alpha Innotech AlphaImager 2200 and/or with 652 nm light excitation on a Li-Cor Odyssey CLx imaging system (LI-COR).

SMARCA4 (BRG1) levels of the HA-purified BAF complex purifications were normalized via BCA protein quantification and immunoblotting analyses. Purified canonical BAF complexes were diluted for final reaction concentration of 10 ng/μL in REAA buffer (20 mM HEPES, pH 8.0, 50 mM KCl, 5 mM MgCl₂) containing 0.1 mg/mL BSA, 1 μM DTT, 20 nM nucleosomes (EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, EpiCypher). The REAA mixture was incubated at 30 or 37°C for 10 minutes, and the reaction was initiated by addition of 1-2 mM ATP (Ultrapure ATP, Promega) and 0.005 U/μL DpnII Restriction Enzyme (New England Biolabs). The REAA reaction mixture was quenched with 20-24 mM EDTA and placed on ice. Proteinase K (Ambion) was added at (100 μg/mL) for 30-60 minutes, followed by either AMPure bead DNA purification and D1000 HS DNA ScreenTape Analysis (Agilent)

SMARCA4 (BRG1) levels of the ammonium sulfate nuclear extracts were normalized via BCA protein quantification and Silver Stain analyses for HA-SS18 and HA-SS18-SSX conditions. Protein was diluted for final reaction concentration of 150 ug/mL in REAA buffer (20mMHEPES, pH 8.0, 50mMKCl, 5mMMgCl2) containing 0.1 mg/mL BSA, 1 mMDTT, 20 nM nucleosomes (EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, EpiCypher). The REAA mixture was incubated at 37C for 10 min, and reaction was initiated using 1–2 mM ATP (Ultrapure ATP, Promega) and 0.005 U/mL DpnII Restriction Enzyme (New England Biolabs). The REAA reaction mixture was quenched with 20–24 mM EDTA and placed on ice. Proteinase K (Ambion) was added at (100 mg/mL) for 30–60 min, followed by either AMPure bead DNA purification and D1000 HS DNA ScreenTape Analysis (Agilent) or mixing with GelPilot Loading Dye (QIAGEN) and loading onto 8% TBE gel (Novex 8%TBE Gels, Thermo Fisher). TBE gels were stained with either SYBR-Safe (Invitrogen) or Syto-60 Red Fluorescent Nucleic Acid Stain (Invitrogen), followed by imaging with UV light on an Alpha Innotech AlphaImager 2200 and/or with 652 nm light excitation on a Li-Cor Odyssey CLx imaging system (LI-COR).