GSM7815570: iFoxA1, mES+24hrs, antiFoxA1-ChIP-exo, rep2; Mus musculus; OTHER
Lysis was performed in Farnham cell lysis buffer for 20mins at 4°C. At the 10-min mark, cells were pushed through a 25-gauge needle five times to enhance cellular lysis. Nuclei were then isolated by pelleting at 10,000 rpm for 3 min. Nuclei were resuspended in RIPA buffer (25 million cells to 1 mL of buffer) for an additional 20 min at 4°C and then pelleted again at 10,000 rpm for 3 min. Disrupted nuclei were then finally resuspended in 1× PBS (25 million cells to 1 mL of buffer) and sonicated for 10 cycles (30-on/30-off) in a Diagenode pico. Solubilized chromatin was pre-cleared by centrifugation at maximum speed for 15 min at 4°C before incubating with 10 μL pAG Dynabeads, preloaded with 3 μg of antibody overnight, then sequentially processed through A-tailing, first adapter ligation, Phi29 fill-in, lambda exonuclease digestion, cross-link reversal, second adapter ligation, and PCR followed by size selection for final high-throughput sequencing.