Nucleosome sliding assay

Nucleosome sliding assays were performed as previously described (Sinha and Peterson, 2009). Briefly, end positioned mononucleosomes were reconstituted onto a 255 bp ³²P-labelled 601 DNA fragment. Mononuleosomes (5 nM) were incubated with purified INO80-C and 2 μM ATP in buffer A (10 mM Tris-HCl pH 8.0, 70 mM NaCl, 5 mM MgCl₂, 0.1 mg/mL 1 BSA and 1 mM DTT) for 15 min at 30°C. The reactions were quenched with 5% glycerol and 1 mg/mL salmon sperm DNA, followed by 5 min incubation at 30°C. The products were resolved by Native-PAGE on 5% polyacrylamide 1xTBE gels. Gels were dried and products visualized using PhosphorImager (Molecular Dynamics).