Hd-rR liver ChIP-seq input for H3K4me2
Cells were dissociated using a 21G needle and fixed with 1% formaldehyde for 10 min at RT then quenched by adding Glycine (200 mM final). After washing with PBS containing 20 mM Na-butylate, complete protease inhibitor (Roche) and 1 mM PMSF, cell pellets were suspended by Lysis buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1 % SDS, 20 mM Na-butylate, complete protease inhibitors, 1 mM PMSF), sonicated for 10 times at vol. 5 of sonifier (Branson), and centrifuged for collecting chromatin lysates. The chromatin lysates were diluted with ChIP RIPA buffer (10 mM Tris-HCl pH8.0, 140 mM NaCl,1 mM EDTA, 0.5 mM EGTA, 1 % Triton-X100, 0.1 % SDS, 0.2 % Na-Deoxycholate, 20 mM Na-butylate, complete protease inhibitors, 1 mM PMSF) and rotated with antibody/protein A Dynabeads complex for over night at 4 oC. Immunoprecipitated materials were washed three times with ChIP RIPA buffer and once with TE buffer, followed by elusion with Lysis buffer at 65 oC over night. Eluted samples were treated with RNase A for 2 h at 37 oC and proteinase K for 2 h at 55 oC, and DNA was purified by Phenol/Chloroform extraction and ethanol precipitation. Libraries were prepared using the TruSeq DNA sample prep kit (version 2; illumina).
UTGSFSCB-ML
Hd-rR liver ChIP-seq input for H3K4me2