GSM8283817: HL-60 H3K27ac CM-444; Homo sapiens; OTHER
CUT&RUN was performed with the kit CUTANA ChIC/CUT&RUN (Epicypher, 14-1048) following the manufacturer's instructions. Briefly, 0.5 million of HL-60 cells were harvested and resuspended in 100ul wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, supplemented with Protease Inhibitor EDTA-Free tablet (Roche 11836170001)]. Activated Concanavalin A was incubated with cells at room temperature for 10min to let the cells bind to the beads. For each target protein factor, 0.5μg of BRD4 CUTANA CUT&RUN antibody (Cat No 13-2003, Epicypher), Acetyl-Histone H3 (Lys 9) (C5B11) rabbit monoclonal antibody diluted 1:50 (Cat No 9649T, Cell Signaling) or Acetyl-Histone H3 (Lys 27) (D5E4) XP rabbit monoclonal antibody diluted 1:100 (Cat No 8173T, Cell Signaling) was added to each sample and incubated in the antibody buffer (wash buffer +0.01% Digitonin and 2 mM EDTA) overnight at 4°C. Isotype control IgG was used as a negative control. The beads were then washed twice with digitonin buffer [wash buffer +0.01% Digitonin], and 2.5 μL pAG-MNase was added to each sample. After ten minutes of incubation at room temperature, excessive pAG-MNase was washed out by a two-time digitonin buffer wash. Then targeted chromatin was digested and released from cells by 2 h of incubation with the presence of 2 mM CaCl2 at 4°C. After incubation with stop buffer [340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL Glycogen] for 10 min at 37ºC the supernatant containing CUT&RUN-enriched DNA was purified using the CUTANA DNA Purification kit (Epicypher). Illumina sequencing libraries were prepared from 6 ng of purified CUT&RUN DNA using NEBNext UltraTM II DNA Library Prep Kit for Illumina, following the manufacturer's instructions. For BRD4, some modifications were added following the protocol of Nan Liu (Harvard University; https://dx.doi.org/10.17504/protocols.io.wvgfe3w).