GSM3660531: RPMI8226_IKZF1; Homo sapiens; ChIP-Seq
IKZF1 ChIP-seq was performed using 20 x 106 cells, which were cross-linked for 20 min at room temperature with a final concentration of 1% formaldehyde solution (5mM Hepes - KOH pH 7.5; 10mM NaCl; 100 M EDTA pH 8; 5 M EGTA pH 8; 1% formaldehyde) followed by a 5 min quench with 125 mM glycine. Cross-linked cells were washed twice in PBS. 100ul of Dynal protein G magnetic beads (Sigma) were washed 3X for 5 min at 4 C in 0.5% BSA in PBS prior to blocking for >5 hrs with 0.5% BSA in PBS. Magnetic beads were bound with 10  mg of IKZF1 antibody (GeneTex, GTX129438). Cross-linked cells were lysed with lysis buffer 1 (50 mM HEPES pH 7.3; 140 mM NaCl; 1 mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100; protease inhibitors) and resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100) prior to sonication on a E210 (Covaris). Sonicated lysates were incubated overnight at 4C with Dynal magnetic beads bound with antibody. Beads were washed two times with sonication buffer, one time with sonication buffer with 500 mM NaCl, one time with LiCl wash buffer (10 mM Tris pH 8; 1 mM EDTA; 250 mM LiCl; 1% NP-40) and one time with TE + 50 mM NaCl. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS) at 65C. Cross-links were reversed overnight at 65C and RNA and protein were digested using first RNase A and then Proteinase K at 37C. DNA was purified with phenol chloroform extraction and ethanol precipitated prior to library preparation Rubicon ThruPLEX DNA-seq (Rubicon Genomics). Libraries were sequenced to a depth of at least 50 x 106 reads on an Ion Proton (Thermo Fisher) sequencer.
GEO Accession
GSM3660531