GSM4875336: RIF1_HCT_WT_r2_CutnRun; Homo sapiens; OTHER
Cells were washed and bound to Concanavalin-A-coated magnetic beads, and permeabilized in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and one Roche Complete protein inhibitor tablet per 50 mL) containing 0.03% digitonin and 20 mM EDTA (Dig-wash). Antibody was added to a final concentration of 1:100 and incubated overnight at 4°C. Cells were washed with Dig-wash buffer and incubated with Protein-A-MNase (pA-MN) for 1 hour at 4°C. Cells were washed three times with Dig-wash buffer to remove unbound pA-MN before placing cells in an ice-cold block and activating cleavage with the addition of CaCl2 to 2mM final concentration. Cleavage was stopped by the addition of 2x Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/mL glycogen, 5 μg/mL RNase A), and fragments were released by 30 min incubation at 37°C. Supernatant was recovered and DNA was purified with phenol:chloroform extraction and precipitation with ethanol, before being used as input for library preparation. Sequencing libraries were prepared from DNA fragments as described in (PMID: 28079019) but without size-selection, following the KAPA DNA polymerase library preparation kit protocol
GEO Accession
GSM4875336