Nucleosome Core Particle Reconstitution

NCPs were assembled essentially as described^{15 (link),56 (link)}. Briefly Human H2A, H2A K119C, H2B, H3.3, H3.1 C96SC110A and H4 were over-expressed in E.coli and purified from inclusion bodies. Histones were refolded from denaturing buffer through dialysis against 10 mM Tris-HCl pH 7.5, 2 M NaCl, 5 mM beta-mercaptoethanol, 1 mM EDTA buffer, and octamers were purified by size exclusion chromatography over a Superdex-200 column (GE Healthcare). DNA fragments for wrapping NCPs (171-bp Widom 601 DNA, 145-bp D02 DNA or D02 DNA appended with biotin and fluorophores) were generated by PCR using Pfu polymerase and HPLC-grade oligonucleotides (IDT). PCR products generated in 96-well plates (384 × 100 μl) were pooled, filtered and purified on a ResorceQ column as described^{15 (link)}. NCPs were assembled by salt dialysis as described^{15 (link),30 (link),56 (link)} and heat repositioned at 37 °C for 30 min. D02 containing NCPs were further purified using a PrepCell apparatus with a 5% polyacrylamide gel (BioRad).