GSM8001258: H4-glob MNase; Escherichia coli; MNase-Seq
Plasmids were prepared using Qiagen Large-construct kit (cat. # 12462) according to the manufacturer's instruction, and then cleaned with phenol chloroform extraction, precipitated, and resuspended in 10 mM Tris pH 8.0, 0.2 mM EDTA. Reconstituted BRCA1-TAR/YAC/BAC arrays were assembled using Xenopus histone octamers with wild-type (W-T) and H2A/H4 N-tailless histones obtained from Histone Source, Protein Expression and Purification Facility at Colorado State University. Reconstitution was performed as described earlier (31). Briefly, core histone octamers and purified DNA were combined in a final mixture containing 2 M NaCl and 1 mM PMSF. Samples were kept on ice for 1 hour following dialysis in 3500 MWCO membranes against buffer (2 M NaCl, 10 mM Tris, 0.2 mM EDTA, 0.1% NP-40, 5 mM β-mercaptoethanol, pH=8.0) for 2 h. Samples were transferred to 1.5 M NaCl buffer and dialyzed for 2 h, followed by dialysis against 1 M NaCl and 0.75 M NaCl buffers, 3 hours each, and 0.5 M NaCl buffer overnight (all other buffer component were kept constant). Reconstitutes were next dialyzed against a buffer containing 5 mM NaCl, 10 mM Tris, 0.2 mM EDTA, 0.1% NP-40, 5 mM β-mercaptoethanol, pH 8.0, for 3 h. Samples were dialyzed in the buffer with 5 mM NaCl, 10 mM Tris, 0.025% NP-40 and no EDTA for additional 3 h. Reconstituted chromatin (1 µg in 60 µl) in buffer containing 50 mM NaCl, 10 mM Tris 0.025% NP-40, 20% Ficoll, and 0.7 mM CaCl2 was digested with 0.2 units MNase  for 10 min at 20°C. Reaction was stopped by addition of 20 mM EDTA and cooled on ice. Digested samples were treated with SDS and proteinase K for 2 h at 55°C and cleaned with Promega Wizard SV Gel and PCR Clean-Up System (cat. # A9282). Nucleosomal DNA fragments were subjected to paired-end sequencing on Illumina platform.