GSM8820043: anti-P53_CUT-RUN_mTEChi_Rep1; Mus musculus; OTHER
500,000 cells were washed three times in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× EDTA-free protease inhibitor cocktail, Roche), then bound to Concanavalin-A beads (Bangs Laboratories) according to the manufacturer's instructions. Cells were incubated with 1:100 dilution of anti-BAF155 antibody (G. Crabtree) for 2 h or overnight at 4 °C in permeabilization buffer (1× permeabilization buffer, eBioscience, 0.5 mM spermidine, 1× EDTA-free protease inhibitor cocktail, 2 mM EDTA). The sample was then incubated with 700 ng ml−1 protein A-MNase (S. Henikoff) in permeabilization buffer at 4 °C for 1 h. Digestion was performed in 0.5× permeabilization buffer supplemented with 2 mM CaCl2 at 4 °C for 1 h. The reaction was stopped by adding 2× Stop buffer (final concentration 100 mM NaCl, 10 mM EDTA, 2 mM EGTA, 20 μg ml−1 glycogen, 25 μg ml−1 RNase A, Thermo Fisher Scientific) and the sample was incubated at 37 °C for 20 min. Protein in the sample was then digested in 0.1% SDS and 250 μg ml−1 proteinase K (New England Biolabs) for 2 h at 56 °C, shaking gently. CUT&RUN fragments were purified using phenol chloroform extraction. CUT&RUN libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina coupled with NEBNext Multiplex Oligos for Illumina (New England Biolabs) with modifications optimized for small fragments as detailed in https://doi.org/10.17504/protocols.io.wvgfe3w. Paired-end, dual-index sequencing was performed on the Illumina NextSeq 500 system.