Plasmids encoding human histones H2A, H2B, H3, and H4 were obtained from K. Luger. Plasmids were transformed into Escherichia coli BL21 Star (DE3). Bacteria were cultured in 2× TY medium at 37°C, 200 rpm until the OD600 (optical density at 600 nm) reached 0.4, and the protein expression was induced with isopropyl-β-d-thiogalactopyranoside (IPTG) (Golden Biotechnology) at a final concentration of 0.4 mM for 3 hours. Bacteria were pelleted down by centrifugation and resuspended in wash buffer [50 mM tris-HCl (pH 7.5), 100 mM NaCl, 1 mM benzamidine, 1 mM β-mercaptoethanol]. The histone purification protocol was established as previously described (60). In brief, cell pellets were lysed and inclusion bodies were dissolved in 7 M guanidine HCl. The denatured histones were purified by size exclusion and ion exchange, and refolded in refolding buffer [2 M NaCl, 10 mM tris-HCl (pH 7.5), 1 mM EDTA, 10 mM β-mercaptoethanol]. The refolded histones were further dialyzed to histone storage buffer [300 mM NaCl, 20 mM tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT].

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