GSM6528174: C002_T3 H3K27ac ChIPseq; Homo sapiens; ChIP-Seq
50 mg of snap frozen liver tissue was excised and pulverized into fine powder in liquid nitrogen. The samples were washed twice in cold 1X PBS with protease inhibitor, cross-linked with formaldehyde (1%) for 10 min at room temperature, quenched with glycine (125mM) for 5 min, and rinsed twice with cold 1X PBS. Lysis was performed for 30 minutes in cold lysis buffer (50mM HEPES-KOH, pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X, 0.1% Sodium deoxycholate, 0.1% SDS, Takara 1X protease inhibitors) to release nuclei. Pellets were centrifugated at 2,500G for 10 min at 4ºC to collect the nuclei pellet. Nuclei was sonicated in nuclei lysis buffer (50mM HEPES-KOH, pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X, 0.1% Sodium deoxycholate, 1% SDS, Takara, 1x protease inhibitor) using the Bioruptor sonicator to obtain chromatin fragments between 200 to 500 bp. The fragmented chromatin was immunoprecipitated overnight with 5 µg of H3K27ac antibody (catalogue #: ab4729, Abcam) and 50 µl of protein G beads (Invitrogen). Elution of DNA was done in 200 µl of elution buffer (50mM Tris-HCI, pH7.5 and 10mM EDTA), followed by decrosslinking which was performed at 65ºC overnight. Library preparation was performed using eluted ChIP DNA with NEB ultra II library preparation kit, according to manufacturer's protocol. 10-12 PCR cycles were performed using indexed primers and libraries size between 300 to 500 bp were selected. The libraries were sequenced at Macrogen using 2 x 150 bp read lengths.