title: GSM3463219: U87_H3K27Ac; Homo sapiens; ChIP-Seq
library_construction_protocol: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Chromatin from formaldehyde-fixed cells (SF763 PTEN-KO cell and U87 cells, 10 x106 cells for CHD1 antibody and 1x106 cells for H3K4me3 antibody) were cross-linked using 1% paraformaldehyde for 10 minutes and reactions were quenched by 0.125 M glycine for 5 minutes at room temperature. Cells were lysed with ChIP lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], 140 mM NaCl, 1% Triton X-100, 0.2% SDS, 0.1% deoxycholic acid) for 30 minutes on ice. Chromatin fragmentation was performed using a Diagenode Bioruptor®Pico sonicator (30 secs on and 30 secs off for 45 cycles) to achieve a DNA shear length of 200 to 500 basepairs. Solubilized chromatin was then incubated overnight with their respective antibody-Dynabead (Life Technologies) mixture (H3K27ac antibody: Abcam #ab4729). Immune complexes were then washed 3 times with RIPA buffer, once with RIPA-500 (RIPA with 500 mM NaCl), and once with LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% deoxycholic acid). Elution and reverse crosslinking were performed in direct elution buffer (10 mM Tris-Cl [pH 8.0], 5 mM EDTA, 300 mM NaCl, 0.5% SDS) with proteinase K (20 mg/ml) at 65°C overnight. Libraries were prepared using NEBNext Ultra DNA Library kit (E7370) from NEB.