GSM6268812: K562 e3i&e7i_H3K27ac ChIP-seq; Homo sapiens; ChIP-Seq
Cells were fixed with 1% Formaldehyde in culture medium at room temperature for 10 minutes. Quench the reaction by adding 250 mM Glycine, then wash cells twice with cold PBS. Three million fixed cells were used for each H3K27ac ChIP-seq assay. Resuspend cells with 1 x TE buffer plus 1 x protease inhibitor cocktail and 1 mM PMSF. Chromatin was sheared by sonication on a Diagenode Bioruptor Pico device at 4 °C for 18 cycles with 30 seconds on and 30 seconds off. Adjust chromatin solution to 1 x RIPA buffer (1 x TE, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100) plus 500 mM NaCl. After centrifuging at 13, 000 rpm for 10 min at 4 °C, chromatin supernatant was collected and 10% of it was saved at input. Mix 2 mg H3K27ac antibody (Abcam, ab4729) with 20 mL Dynabeads Protein A beads (ThermoFischer Scientific, Cat. 10001D), then incubate at room temperature for 1 hour with rotation. Wash beads once with 1 x PBS, then add chromatin solution to beads, and incubate at 4 °C overnight with rotation. Wash beads three times with RIPA buffer plus 500 mM NaCl, then three times with RIPA buffer plus 1 M NaCl, then three times with LiCl buffer (1 x TE, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate), and finally three times with 1 x TE buffer. Elute DNA and reverse crosslinking with protease K digestion and incubating at 65 °C for over 6 hours. DNA was purified with MinElute Reaction Cleanup Kit (QIAGEN, Cat. 28206). Repair DNA ends with End-It DNA End-Repair Kit (Lucigen, Cat. ER0720), then perform A-tailing with Klenow Fragment (3' -> 5' exo-) (NEB, Cat. M0212S) provided with dATP, then followed by adapter ligation with T4 DNA ligase (NEB, Cat. M0202S). Finally, the library was indexed and amplified by PCR, and DNA fragments between 200 bp and 600 bp were purified and sequenced.