GSM7688873
Cells were fixed in 1% formaldehyde and after washing in PBS, left for 1 hour in 50 mM DTT. After quenching with 100mM NEM (N-ethylmaleimide) and washing in PBS, cells were incubated with DpnII overnight at 37°C. After restriction digestion, cells were incubated for 4 hours at 23°C with the Klenow DNA polymerase I to fill in the 5' overhang and incorporate biotin-14-dATP followed by chromatin ligation for 4 hours at 16°C with T4 DNA ligase (Invitrogen). Then cells were left overnight in 400 ng/ml proteinase K and 0.5% SDS at 60°C overnight to reverse cross-linking. After DNA purification and precipitation, DNA was sonicated to 200-300 bp fragments and an additional size selection was done with Ampure beads. To repair DNA ends after sonication, T4 DNA polymerase (NEB) and Klenow DNA polymerase (NEB) were used together with T4 polynucleotide kinase (NEB). Biotinylated ligation products were pulled down using streptavidin beads (Invitrogen) followed by A-tailing and adaptor ligation for library construction