GSM4969604: SF7761-H3K27AC Rep 1; Homo sapiens; ChIP-Seq
Briefly, 30-50 million cells were crosslinked using 1% formaldehyde for 10 minutes and quenched with 0.125M glycine for 5 minutes at room temperature. Cells were then rinsed, harvested, and resuspended in extraction buffer for ten minutes at 4°C, then centrifuged at 1,350xg for five minutes at 4°C. Cell pellet was resuspended and transferred to a one mL milliTUBE (520135, Covaris). After sonication (Covaris E220 ultrasonicator), samples were spun down and supernatant containing chromatin was collected. Chromatin was de-crosslinked for three hours at 65°C and DNA was extracted (28104, Qiagen). 10% sample was saved as input. Chromatin was incubated with primary antibody overnight at 4°C. The following day, protein A/G agarose beads (sc-2003, Santa Cruz Biotech) were added to the samples and incubated for 3 hours. Beads were centrifuged and washed with one mL of RIPA buffer four times. The beads and the ten mL input were eluted for 30 minutes at 65°C then spun down. Supernatant was de-crosslinked overnight 65°C. DNA was extracted and submitted for library preparation. ChIP-Seq libraries were prepared with the KAPA Library preparation kit (KK8234, Kapa Biosystems) and NETflex DNA barcodes (514104, Bioo Scientific). A total of ten mg DNA was used as starting material for input and immunoprecipitation samples. Libraries were amplified with a thermocycler for 13 cycles. Post-amplification libraries were size-selected at 250-45bp in length using the Agencourt AMPure XP beads (A63881, Beckman Coulter).