We gel-purified the 149-bp and 249-bp DNA fragments, used them for nucleosome reconstitution by octamer exchange at 1:3 DNA:chromatin ratio17 (link). The hexasomes were reconstituted using chicken erythrocytes core histones by dialysis from 2 M NaCl48 (link).
To obtain 603-42 and 603-49 templates for E. coli RNAP we mutated the original 603 template to replace four or six nucleotides in DNA and allow stalling of RNAP at the +42 or +49 positions within the 603 nucleosome, respectively. We amplified the nucleosome positioning sequences by PCR, digested by TspRI (NEB) and ligated through the TspRI site to a T7A1 promoter-bearing fragment30 (link). Ligated products were re-amplified with one 5′-end-labeled primer, gel-purified, and assembled into nucleosomes. We reconstituted nucleosomes on the DNA templates by histone octamer transfer from chicken -H1 erythrocyte donor chromatin17 (link).
To obtain 603-42 and 603-49 templates for E. coli RNAP we mutated the original 603 template to replace four or six nucleotides in DNA and allow stalling of RNAP at the +42 or +49 positions within the 603 nucleosome, respectively. We amplified the nucleosome positioning sequences by PCR, digested by TspRI (NEB) and ligated through the TspRI site to a T7A1 promoter-bearing fragment30 (link). Ligated products were re-amplified with one 5′-end-labeled primer, gel-purified, and assembled into nucleosomes. We reconstituted nucleosomes on the DNA templates by histone octamer transfer from chicken -H1 erythrocyte donor chromatin17 (link).
