GSM5155153: MV411_DMSO_Input_24h; Homo sapiens; ChIP-Seq
25 million (for histone marks) to 100 million AML cells (for IRF2BP2) per condition were crosslinked with 1% formaldehyde in cell culture media for 10 minutes and then quenched with 0.125 mM glycine. Cells were lysed in Nuclei EZ Isolation buffer (Sigma EZ Prep Nuclear Isolation Kit, Cat No. NUC-101) supplemented with protease inhibitor. Chromatin was sheared using a Covaris instrument. Subsequently, 0.5% of the lysate was removed as the input control. The remaining lysate was incubated with 30 ul (for histone marks) or 100 l (for IRF2BP2) of protein G dynabeads (Thermofisher) and 5 ug of H3K27Ac (ab4729 abcam) or 10 ug of IRF2BP2 antibody at 4 degrees C overnight (antibodies and beads were incubated together for 30 minutes at 4 degrees C prior to the addition of cell lysate). The precipitated lysate was then washed sequentially in two washes of each of the following buffers: ice cold low salt buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 nM EDTA, 1% Triton-X100, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton-X100, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% desoxycholic acid, 1% IGEPAL CA-630) and TE buffer (10 mM Tris-HCl ph8.1, 1 mM EDTA). Samples were eluted in elution buffer (1x TE pH 7.4, 1% SDS, 150 mM NaCl, 5 mM DTT). RNase A, proteinase K, and 0.2 M NaCl was added to the elution buffer and samples were incubated at 65 degrees Celsius for at least four hours to reverse cross-linking. DNA was purified using AMPure XP beads (Agencourt). ChIP-seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200. Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq.