On 15-cm plates, C4-12/Flag.ERβ cells were treated with 10nM E2 for 1 hour then crosslinked with 1% formaldehyde. Cells were lysed with 1mL of ChIP-lysis buffer (50mM Tris pH 7.4, 100mM NaCl, 0.1% SDS, 1% Triton-X, 0.5% NP40, PICIII) and sonicated for 10 cycles, each of which was 10 seconds. The lysate was collected and incubated with 30uL Protein G Plus-Agarose (Santa Cruz) preimmuned with 10ug anti-Flag M2 antibodies (Sigma) to capture protein-DNA complex overnight in ChIP-lysis buffer. The Protein G beads were washed once with ChIP-wash buffer I (20mM Tris pH 8.1, 150mM NaCl, 0.1% SDS, 1% Triton X, 2mM EDTA), once with ChIP-wash buffer II (20mM Tris pH 8.1, 500mM NaCl, 0.1% SDS, 1% Triton X, 2mM EDTA), once with ChIP-wash buffer III (10mM Tris pH 8.1, 250mM LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA), and twice with TE buffer. The protein-DNA complexes were eluted with ChIP-elution buffer (100mM NaHCO3, 1% SDS). The crosslinking was reversed by incubating samples at 65oC overnight. After treatment of RNase A and Proteinase K, the inputs and immunoprecipitated samples were extracted once with phenol/chloroform, once with chloroform, and precipitated in ethanol. The genomic DNA precipitate was suspended in 20uL nuclease-free water. Illumina ChIP-seq kit
GEO Accession
GSM1168432