GSM6621041: induced MCF10A YAP5SA cells control; Homo sapiens; OTHER
For CUT&RUN-seq, 500000 cells were harvested and washed twice with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM spermidine containing protease inhibitor) and captured with 20 μl conacavallin A magnetic beads (Polyscience). Cells were resuspended with antibody buffer (wash buffer with 0.005% digitonin and 2 mM EDTA) and incubated with 2 μg of YAP-antibody (Novus Biologicals, NB110-58358) for 2 hours at room temperature. Samples were then washed twice with wash buffer containing 0.005% digitonin and incubated with 700 ng/ml of purified protein-A/G-MNase fusion (pA/G-MNase) on a shaker at 4°C for 1 h followed by two more washes in digitonin wash buffer and once in low salt rinse buffer [20mM HEPES pH7.5, 0.5mM Spermidine, 0.005% Digitonin]. To activate protein A-MNase, incubation buffer (3.5mM HEPES, 10mMCaCl2, 0.005% digitonin) was added and the DNA was digested for 30min on ice. The incubation buffer was discarded and the reaction was stopped by resuspension in STOP buffer (170 mM NaCl, 20 mM EDTA, 0.005% Digitonin, 50μg/ml RNaseA, 25μg/ml Glycogen). The protein-DNA complex was released by incubation at 37°C for 30min, the supernatant was transferred to a fresh tube and then digested by proteinase K at 50°C for 1 hours. DNA was extracted by ethanol precipitation. Libraries were made with 6 ng of CUT&RUN DNA fragments using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S). The end prep was performed at 20°C for 30min followed by 50°C for 1h. The adaptor was used at a concentration of 0.5 μM. 15 PCR cycles were performed at the following conditions: Initial denaturation: 98°C for 30sec. Denaturation 98°C for 10sec; Annealing/Extension 65°C for 10sec and final Extension 65°C for 5min. Library quality was analyzed using the Fragment Analyzer (Advanced Analytical). Libraries were sequenced on the NextSeq 2000 platform (Illumina).