Pag mnase enzyme

CUT&RUN was performed as previously described^{98 (link)}. 5x10⁵ cells were washed twice in PBS and resuspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 x Roche complete EDTA-free protease inhibitor), followed by immobilization on concanavalin A magnetic beads activated in Binding Buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂). Bead-bound cells were then incubated with antibody in Antibody Buffer (Wash Buffer supplemented with 2 mM EDTA and 0.05% Digitonin) for 2 hours at room temperature. Following incubation, beads were washed twice with Digitonin Buffer (Wash Buffer supplemented with 0.05% Digitonin) and incubated 1 hour at room temperature in Digitonin Buffer with pAG-MNase enzyme (Cell Signaling). Next, beads were washed twice with Digitonin Buffer and MNase was activated by adding CaCl₂ (50 μL of Digitonin Buffer supplemented with 4mM CaCl₂) and incubated for 1 hour at 37°C. DNA digestion was stopped by the addition of 2X Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.1% Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen) and incubated at 37°C for 1 hour. DNA fragments were collected in the supernatant and purified using MinElute PCR purification kit (Qiagen). Sequencing libraries were prepared using Rubicon ThruPLEX DNA-seq kit, followed by sequencing on the NextSeq500 using 75bp paired-end chemistry.