GSM5532081: ChIP-seq_Input_HMEC_rep1; Homo sapiens; ChIP-Seq
(ChIP-seq) The cells were washed twice with PBS and incubated with 1% formaldehyde for 10 min at RT. The crosslinking was quenched with 125 mM glycine for 5min at RT, and the cells were harvested with cold PBS and suspended in SDS lysis buffer (50mM Tris-HCl, pH 8.0, 1% SDS, 10mM EDTA). Following the suspended chromatin was sheared using a ultrasonicator (Covaris S220) and incubated overnight with the relevant antibodies against anti-CTCF (Millipore; 07-729), anti-H3K27ac (Abcam; ab4729) and Protein A, G sepharose (GE Healthcare; 17-1279-03 and 17-0618-05) at 4°C with agitation. The Immune complex was washed in the order of low-salt wash buffer (20mM Tris-HCl, pH8.0, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA), high-salt wash buffer (20mM Tris-HCl, pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and LiCl wash buffer, and finally, washed twice with TE buffer. Following the washed immune complexes were eluted with elution buffer (1% SDS, 0.1M NaHCO3). Finally the immune complexes were eluted with elution buffer (1% SDS, 0.1M NaHCO3) and de-crosslinked overnight at 68°C. The immunoprecipitated DNA was treated with proteinase K and RNase A and phenol-chloroform-isoamyl alcohol precipitation. (ChIP-seq) The ChIP-seq libraries were prepared using a Accel-NGS 2S Plus DNA Library Kit (SWIFT; 21024)