GSM8311107: IMR90_ATRX_KO_HIRA-KD_Rep3_CR_H33; Homo sapiens; OTHER
Cells were pelleted, washed with DPBS, and nuclei were extracted following protocol described in Methods section of the publication. Extracted nuclei were bound to ConA lectin-coated beads, blocked with buffer containing EDTA to chelate any biological reactions, washed, and then incubated with either a primary antibody solution containing an anti-H3.3 antibody (Active Motif Cat: 91191, Lot: 25820004) or a no antibody negative control for 1hr at RT with rotation. Next, nuclei with bound primary antibody were incubated with pA/G-MNase for 30 minutes at room temperature with rotation.  pA/G-MNase activity was initiated by addition of calcium and digestion controlled with samples on ice water bath for 30minutes. Following digestion period, fragments were solubilized and used as input to the library build protocol to prepare deep sequencing libraries. CUT&RUN libraries were amplified by high-fidelity PCR with barcoded NEB indices.