GSM8375132: Flag_input_HCT116; Homo sapiens; ChIP-Seq
Cells were cross-linked by incubating them initially in Gold (Diagenode), and then they were further crosslinked in DPBS (Gibco, Cat #14190094)  supplemented with 1% formaldehyde (Sigma-Aldrich; Cat #252549) for 10 min rocking at room temperature. Cross-linking was stopped using 125 mM Glycine (Sigma-Aldrich, Cat #G8790) rocking for 5 minutes at room temperature. Cell nuclei were isolated by incubating cell pellets in a buffer containing 5mM Pipes pH 8, 85mM KCl and 0.5% NP40, and later incubated on ice for 10 minutes followed by centrifuge at 4000 rpm for 5 minutes at 4ºC. Nucleus were lysed in a buffer containing 1% SDS, 10mM EDTA and 50 mM Tris-HCl pH 8.1 and incubated on ice for 10 min. Samples were sonicated 25 cycles (30´´on/30´´off)  and SDS was removed by incubating the sonicated sample on ice for 1 hour and later centrifuged for 15 at 4ºC and 16000g. 60μg of chromatin was incubated with 10 μg of ANTI-FLAG® M2 (Sigma-Aldrich, Cat #F1804). Samples were pulled down by a 50/50 mix of Dynabeads A (Invitrogen, Cat #10002D) and G (Invitrogen, Cat #10003D). Samples were washed 4 times in 0.1% SDS, 1% Triton-X, 2mM EDTA, 20mM Tris-HCl pH 8 and 150mM NaCl; followed by 1 wash in TE pH 8.  Samples were then eluted in 1% SDS and 100mM NaHCO3. Samples were descrosslinked by incubating for 16 hours (or overnight) at 65ºC shaking at 450 rpm, and were treated with Proteinase K for 1h at 45ºC. DNA was purified using QIAquick PCR Purification Kit (Qiagen, Cat #28106), following the manufacturer's instructions. Libraries prepared according to manufacturer's instructions (SE 50bp reads)