GSM2758961: INPUT_Hp; Homo sapiens; ChIP-Seq
HUVEC cells  fixed and protein‐DNA complexes crosslinked by treatment with 1% formaldehyde, and then quenched with glycine (125mM). Next, cells were washed 3 times with ice‐cold PBS, collected using a cell scraper and resuspended in ice‐cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris‐HCl pH 8.1). DNA was sheared by using a sonicator (Diagenode Bioruptor, Belgium). Samples were diluted 1/10 with dilution Buffer (0.01% SDS, 1.1% Triton X‐100, 1.2 mM EDTA, 16.7 mM Tris‐HCl pH 8.1, 167 mM NaCl) and then precleared with Salmon Sperm DNA/Protein A agarose 50% slurry (Fast Flow, Millipore, 16‐156.) Sample were immunoprecipitated with the antibodies SIN3A (ab3479) at 4°C rocking overnight. Immunocomplexes were recovered by addition of Salmon Sperm DNA/Protein A agarose 50% slurry. Then samples were sequentially washed in Low Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris‐HCl pH 8.1, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris‐HCl pH 8.1, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP‐40, 1% NaDesoxycholate, 1 mM EDTA, 10 mM Tris‐HCl pH 8.1), and twice in TE buffer (10 mM Tris‐HCl pH 8, 1 mM EDTA). Elution of protein‐bound DNA was performed using 500 μl of fresh elution buffer (1% SDS, 0.1 M NaHCO3). Next crosslinking of all samples was reversed by the overnight incubation with 200 mM NaCl at 65°C. Next day, proteins were removed by the addition of 10 μl of 0.5 M EDTA, 20 μl Tris‐HCl pH 6.5 and proteinase K (40 μg/sample). Then immunoprecipitated DNA was purified by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation (UltraPure™  phenol:Chloroform:Isoamyl Alcohol 25:24:1, Invitrogen, 15593‐031). Libraries were prepared according to TruSeq DNA library kit.
GEO Accession
GSM2758961