title: GSM3018484: H3K27ac HiChIP Resistant LSD1i; Mus musculus; OTHER
library_construction_protocol: 5000000 cells were crosslinked with 1% formaldehyde for 10 mins, digested with Mbo1 and ligated to join close proximity fragments, as reported previously. Post ligation, nuclei were lysed using ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8) and sonicated using the Covaris S2 Ultrasonicator (Fill level:10, Duty Cycle:10%, Power: 70W, Burst:200, Time: 8mins-30 secs on, 30 secs off). Sonicated chromatin was then diluted 1:8 in ChIP dilution buffer (1% Triton-X, 0.1% Na-Doxycholate, 90mM NaCl, 10mM Tris-HCl pH 8) before being incubated with 5ug of H3K27ac antibody overnight at 4°C. Antibody-protein complexes were then incubated with Protein A Dynabeads (Thermo Fischer) for at least 2hrs before being washed twice with t low salt buffer (0.1% SDS, 1% Triton-X, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8), once with high salt buffer (0.1% SDS, 1% Triton-X, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8), once with LiCl wash buffer (1% Na-Doxycholate, 1% IGEPAL-CA630, 2mM EDTA, 250mM LiCl, 20mM Tris-HCl pH 8) and once with TE. Enriched complexes were then eluted with elution buffer (1% SDS, 0.1M NaHCO3) for 20mins at 30°C. Reverse crosslinking was performed overnight at 65°C with RNAse A and NaCl. The following day, purified DNA was isolated in TE with Minelute columns (Qiagen) ThruPLEXTM-FD Prep Kit protocol (Rubicon Genomics)
attributes.tag: GEO Accession
attributes.value: GSM3018484