GSM2973421: Healthy Input; Homo sapiens; ChIP-Seq
BECs were treated with hypotonic solution and mild detergent followed by clarification through sucrose gradient to isolate nuclei. Nuclei were treated with micrococcal nuclease (10U) for 10 minutes at 37°C, re pelleted and supernant containing mononucleosomes stored at 4°C. Mononuclesomal fractions were incubated with 4μg of H3K27ac antibody (Abcam, Ab4729) and 25μL Protein G dynabeads (Life Technologies) in modified RIPA buffer over night at 4°C. Bound complexes were washed and eluted and ChIP DNA extracted using phenol:chloroform/ethanol precipitation. Libraries were prepared using half total volume of eluted ChIP DNA and NEBNext® DNA Library Prep Master Mix Set and Multiplex Oligos for Illumina® (New England Biolabs). Library quality was assessed using Bioanalyzer 2100 High Sensitivity DNA Gels (Agilent).