GSM4972033: CHD4_BcBL-1_1_CUT&RUN; Homo sapiens; Human gammaherpesvirus 8; OTHER
Cells were washed with PBS and wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine (Sigma, S2626) and proteinase inhibitor (Roche). After removing wash buffer, cells were captured on magnetic ConA beads (Polysciences), in presence of CaCl2. Beads/cells complexes were washed with digiton wash buffer (0.02% digitonin, 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1x proteinase inhibitor) three times, and incubated with specific antibodies in 250 mL volume. After incubation, unbound antibody was washed by digitonin wash buffer 3 times. Beads were then incubated with the recombinant pAG-MNase, in 250 μl digitonin wash buffer (1.0 mg/mL final concentration) for an hour at 4°C with rotation. Unbound pAG-MNase was removed by washing with digitonin wash buffer 3 times. Pre-chilled 2 mM CaCl2 containing digitonin wash buffer (200 mL) was added to beads and incubate on ice for 30 min. The pAG-MNase digestion was halted by the addition of 200 μl 2× STOP solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/ml RNase A, 50 mg/ml Glycogens). The beads were incubated/shaked at 37°C for 10 min in tube shaker at 500 rpm to release digested DNA fragments from the insoluble nuclear chromatin. The supernatant was collected after centrifuge (16,000 x g for 5 min at 4°C) and place on magnetic stand. DNA was extracted using NucleoSpin kit (Takara). Sequencing libraries were prepared from 3 ng of CUT&RUN DNA with the Kapa HyperPrep Kit (Roche) according to the manufacturer's standard protocol. Libraries were multiplex sequenced (2 x 150bp, paired-end) on an Illumina HiSeq 4000 sequencing system. Other: CUT&RUN