Bacterial expression vectors for histones H2A and H2B were purchased from Addgene (42,634 and 42,630, respectively). Plasmids to express X. laevis H3 (xH3) in pET-3d and xH4 in pET-3a were obtained from Professor Arrowsmith (University of Toronto). Introduction of C110A and K79C mutations in xH3 was performed by site-directed mutagenesis using QuikChange (Stratagene), and the plasmid was sequence-verified. Recombinant histones were purified from E. coli, and modified where indicated, before octamer assembly and subsequent refolding of rNCP with a 151 base pair 601 Widom DNA as previously described (Dyer et al, 2003 (link); Galloy et al, 2021 (link)). Briefly, the histones were purified from inclusion bodies under denaturing conditions on a 5-ml HiTrap SP FF (GE Healthcare) cation exchange column on a next-generation chromatography (NGC, Bio-Rad). Fractions containing the purified histone were pooled and dialyzed three times into 4 liters of water and 2 mM β-mercaptoethanol before lyophilization. The four histones were then unfolded into 20 mM Tris, pH 7.5, 7 M guanidine–HCl, and 10 mM DTT and mixed in equimolar ratios before octamer refolding into 2 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA. Octamers were then purified on a Superdex 200 HiLoad 16/600 size exclusion column (GE Healthcare) and wrapped with the 151 base pair 601 Widom DNA to obtain rNCPs. Native gel analysis was used to validate the quality of the reconstitution.
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