GSM8293936: HCT116, SY5609, pSer7, ChIP; Homo sapiens; ChIP-Seq
Cells were crosslinked with a final concentration of 1% methanol-free formaldehyde for ten minutes while shaking at 145 RPM on an orbital shaker. Cells were quenched with 2.5 M Glycine to a final concentration of 125 mM for 8-10 minutes. Cells were harvested, washed with ice cold PBS (no calcium, no magnesium), and incubated in NRO buffer at a concentration of 80 μL/1 million cells for five minutes twice. Nuclei were pelleted, frozen in liquid nitrogen, and stored at -76°C for future use. Nuclei were thawed on ice for 1.5-3 hours. Once pellets thawed, nuclei were resuspended in 1 mL Covaris Lysis Buffer B + 1x Protease Inhibitors and incubated for 10 minutes at 4°C with nutation. Nuclei were then washed with 1 mL Covaris Wash Buffer C + 1x Protease Inhibitors and incubated for 10 minutes at 4°C with nutation. Nuclei were washed with 500 μL Shearing Buffer D3 + 1x Protease Inhibitors and resuspended in 1 mL of the same solution per ~20 million cells. Nuclei were then aliquoted into AFA millitubes (1 mL/tube) and sheared on the Covaris M220 Focused Ultrasonicator for 12 minutes at 6°C, duty factor 10%, peak power 75, 200 cycles/burst. Sheared chromatin was divided into 750-780 μL aliquots (~15 million cells/aliquot) into pre-chilled Eppendorf tubes, frozen in liquid nitrogen, and stored at -76°C for future use if not immediately proceeding to the IP step. If needed, sheared chromatin was thawed on ice for 2-3 hours. Libraries were prepared using the KAPA Hyperprep Kit. 1 ng of total DNA from each sample was diluted in 50 μL 0.1x TE buffer and subjected to end repair and A-tailing. Following ligation, a 0.8x bead cleanup was performed, and the libraries were amplified 14 cycles.