GSM3415555: RAD21 – 1h 110 mM NaCl + 1h isotonic + triptolide pre-treatment; Homo sapiens; ChIP-Seq
Cells were cross-linked in 1% formaldehyde for 10 min, quenched with glycine for 5 min and harvested in 2 mL of PBS with protease inhibitors. The cells were then lysed, first in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% Nonidet P-40) and then in nuclei lysis buffer (50 mM Tris–HCl pH 8.1, 1% SDS, 10 mM EDTA), after which sonication was performed (15x30 sec, intervals of 30 sec). Subsequently DNA was immunoprecipitated with the indicated antibody O/N at 4 ºC with IP buffer (16.7 mM Tris–HCl pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% TritonX100). Pre-blocked beads (50 uL magnetic (Dynabeads from Invitrogen) or 50 uL of Agarose (Diagenode) beads) were incubated with samples for 1 h at 4 ºC, and were then washed with 2x low salt (20 mM Tris–HCl pH 8, 0.1% SDS, 1% Triton x100, 2 mM EDTA, 150 mM NaCl), 2x high salt (20 mM Tris–HCl pH 8, 0.1% SDS, 1% Triton X100, 2 mM EDTA, 500 mM NaCl), 2x LiCl (10 mM Tris–HCl pH 8, 250 mM LiCl, 1% Nonidet-P40; 1% Na deoxycholate; 1 mM EDTA) and 2x TE (10 mM Tris pH 8, 1 mM EDTA). Beads were eluted with 1% SDS, 0.1 M NaHCO3 for 10 min at 65 ºC and were then de-crosslinked O/N at 65 ºC with NaCl (200 mM). The following day, RNAse A digestions were performed for 2 h at 37 ºC and proteinase K digestions were performed for 1 h at 45 ºC. Finally, DNA was extracted using phenol /chloroform /isoamyl precipitation and was eluted in 30 uL of water. CTCF and Pol II ChIP-seq were performed using Protein A Agarose Beads (Diagenode #C03020002). RAD21 ChIP-seq was performed using Invitrogen M-280 sheep anti-rabbit IgG #112.03D magnetic beads. TruSeq ChIP Library Preparation Kit according to manufacturer specifications.
GEO
GSM3415555