GSM5140845: MOLM13_CUTRUN_CTCF_steadystate_REP1_190522_ATTCCT_S13; Homo sapiens; OTHER
5x105 cells were washed twice in PBS, then once in a wash buffer (20mM HEPES pH 8.0, 150mM NaCl, 0.5mM spermidine) followed by immobilization on Concanavalin A magnetic beads activated in binding buffer (20mM HEPES pH 8.0, 10mM KCl, 1mM CaCl2, 1mM MnCl2) (Polyscience). The immobilized cells were permeabilized by washing in wash buffer supplemented with 0.1% Triton-X100 and incubated with anti-IKAROS antibody (Cell Signaling), rabbit IgG control (Bethyl), anti-CTCF (Cell Signaling) or anti-H3K27Ac (Diagenode) at 4C with addition of EDTA to 2mM. After 2-hour incubation the cells were washed twice and incubated with Protein-A-MNase conjugate at 4C. After 4 hours, the cells were washed twice and MNase was activated by addition of CaCl2 to 20mM. The DNA digestion was stopped by addition of EDTA and EGTA, both to 10mM. DNA fragments were collected in supernatant and purified using 1.8x AMPure XP beads (Beckman Coulter) which were then converted to Illumina compatible libraries using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs) NEBNext Ultra II DNA library prep kit for Illumina