GSM3617908: SKBR-3 TFAP2C (2nd ChIP-Seq sample); Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and TFAP2C-DNA complexes were isolated with antibody. One hundred million cells were cross-linked for 10 min at 37°C using 0.7% formaldehyde, 0.125 M glycine. Cells were washed twice with PBS, re-suspended in lysis buffer and incubated for 5 minutes. Cells were collected by centrifugation and cell pellets were frozen in liquid nitrogen and stored at minus 80°C. Cell pellets were thawed in lysis buffer, collected by centrifugation and cell nuclei were re-suspended in RIPA buffer. Chromatin was sonicated using conditions determined empirically for MCF-7 cells to achieve an optimal fragment length between 100 to 400 bp. After sonication, samples were centrifuged at 20,000 × g for 10 min at 4°C. The supernatant containing cross-linked DNA/histones was diluted with IP dilution buffer to 2 mg/ml. Half the sample was immunoprecipitated with 10 μg of TFAP2C monoclonal antibody SC-12762 (Santa Cruz Biotechnology, Santa Cruz, CA), which was previously shown to be specific for TFAP2C without cross reactivity to TFAP2A, and half with control nonspecific IgG (Upstate, Waltham, MA) with the addition of Dynal sheep anti-mouse Dynabeads and allowed to recognize their antigens overnight at 4°C with rotation. Protein/antibody/DNA complexes were collected magnetically followed by washing and elution. Protein/DNA cross-links were reversed using 200 mmol/L NaCl at 65°C overnight. DNA was treated with Proteinase K and RNase A and was recovered with QIAquick PCR Kit (Qiagen) according to manufacturer's suggested protocol. Purified DNA was quantified by using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE).
GEO Accession
GSM3617908