GSM5949080: OCT4 (CUT&RUN conventional) 1; Mus musculus; OTHER
We performed CUT&RUN as described elsewhere (Patty and Hainer, Nat. Protoc. 16:2633-66, 2021) using recombinant Protein A-micrococcal nuclease (pA-MN). Briefly, we extracted nuclei from ~ 4500000 embryonic stem cells using a nuclear extraction buffer and bound to 500 µl pre-washed lectin-coated concanavalin A magnetic beads (Polysciences). Then, we washed beads in binding buffer. We pre-blocked immobilized nuclei with blocking buffer and washed once in wash buffer. Following this, we incubated nuclei in wash buffer containing primary antibody for one hour at 4°C with rotation. We then incubated in wash buffer containing recombinant pA-MN for 30 min at 4°C with rotation. Using ice-water bath, we equilibrated samples to 0°C and added 3 mM CaCl2 to activate pA-MN cleavage. We then performed sub-optimal digestion, at 0°C for 30 min. Following this, we chelated digestion with 2XSTOP+ buffer (including 1.5 pg MNase-digested Saccharomyces cerevisiae mononucleosome-sized DNA spike-in control). We released and separated genomic fragments after RNase A treatment and centrifugation. Finally, we digested protein using proteinase K, and DNA was purified using PCl extraction, followed by ethanol precipitation. We performed library preparation of 5 ng of CUT&RUN DNA, as per the NEB Ultra II Library Preparation Kit (cat. no. E7645L) manufacturer's protocol.