Neb ultra 2 dna library prep kit

CUT&RUN were performed according to manufacturer’s instructions (EpiCypher CUTANA^™ pAG-MNase for ChIC/CUT&RUN, Cat# 15-1116). In brief, after washing with CUT&RUN wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× Roche Complete Protease Inhibitor), a million of cells were first bound to activated ConA beads (Bangs Laboratories, cat# BP531), followed by addition of anti-H4K20me3 antibody (Abcam, ab9053; 1:100 dilution) and cell permeabilization with the digitonin buffer (CUT&RUN wash buffer plus 0.01% digitonin). After washing in the digitonin buffer, samples were incubated with pAG-MNase, followed by additional washes with digitonin buffer. After the final wash, pAG-MNase activation was induced for DNA digestion by suspending cell samples in the pAG-MNase digestion buffer (digitonin buffer plus 2 mM CaCl₂) and incubation on nutator at 4 °C for 2 h. Solubilized chromatin was released using the stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNase A, 50 µg/ml glycogen) and collected using a PCR cleanup kit (New England BioLabs [NEB] Monarch PCR & DNA Cleanup Kit, cat# T1030). Ten nanogram of the purified CUT&RUN-enriched DNA was used for preparation of multiplexed Illumina libraries using the NEB Ultra II DNA Library Prep Kit according to manufacturer’s instructions (NEB cat#E7103).