title: GSM4809303: ChIP_THP1_sgNegCtrl_KO_aH4K16ac; Homo sapiens; ChIP-Seq
library_construction_protocol: The cells were fixed with 1% formaldehyde for 10 min, after which the reaction was quenched by the addition of glycine to the final concentration of 0.125 M. The fixed cells were washed with PBS and resuspended in SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spun down, resuspended in the immunoprecipitation buffer at 1 ml per 16 million cells (SDS buffer and Triton Dilution buffer (100 mM NaCl, 100 mM TrisHCl pH 8.0, 5 mM EDTA, 5% Triton X-100) mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail from Roche) and processed on a Bioruptor Plus Sonicator (Diagenode) to achieve an average fragment length of 200-300 bps. Chromatin concentrations were estimated using Bradford (Bio-Rad) or BCA assays (Thermo Fisher SCIENTIFIC) according to manufacturer's protocols. The immunoprecipitation reactions were set up in 1 ml of the immunoprecipitation buffer as indicated below and incubated overnight at 4C. The next day, BSA-blocked Fast-flow Protein G Sepharose beads (GE Healthcare) or Protein G Dynabeads (Thermo Fisher SCIENTIFIC) were added to the reactions and incubated for 3 hours at 4C. The beads were then washed three times with low-salt washing buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM TrisHCl pH8.0) and two times with high-salt washing buffer (500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM TrisHCl pH8.0). The samples were then reverse-crosslinked overnight in the elution buffer (1% SDS, 0.1 M NaHCO3) and purified using QIAQuick PCR purification kit (QIAGEN). NEBNext® Ultra™ II for DNA Library Prep