GSM9065468: K562 dBRD9 treatment, pA-Mnase; Homo sapiens; OTHER
Concanavalin-A-coated beads (Polysciences Europe GmbH) were activated in binding buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂). Cells were washed with Wash Buffer (Hepes-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and subsequently immobilized on the activated beads in 50 μl Wash Buffer containing 0.05% Digitonin. pA-MNase or RHΔ-MNase fusion protein was added to the cells and incubated overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin (Millipore), samples resuspended in 100 μl Dig-Wash-Buffer were equilibrated on ice. The activity of the MNase was triggered by adding 2 mM CaCl₂ to the samples for 30 minutes. The reaction was stopped by adding 2× Stop Buffer (68 μl 5 M NaCl, 40 μl 0.5 M EDTA, 20 μl 0.2 M EGTA, 10 μl 5% digitonin, 5 μl 10 mg/ml RNase A). Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16,000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 μg proteinase K. Before library preparation, the DNA was recovered by DNA Purification Buffers and Spin Columns kit (Cell Signaling #14209). NGS library preparation was performed using NEBNext Ultra II DNA Library Prep Kit (NEB, E7103). Libraries were prepared with a starting amount of 1 ng of DNA and were amplified in 12 PCR cycles. Libraries were profiled in a D1000 ScreenTape on a TapeStation 4200 (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies).