title: GSM5949085: OCT4 (CUT&RUN bisulfite) 1; Mus musculus; Bisulfite-Seq
library_construction_protocol: 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 2× STOP+ 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 bisulfite-converted adapter-ligated CUT&RUN DNA using Zymo Research EZ DNA Methylation Kit (Zymo, cat. no. D5001) following the alternative protocol for the Illumina Infinium Methylation Assay. Briefly, we added 5 µl of M-Dilution buffer to purified adaptor-ligated DNA, and adjusted total sample volume to 50 µl with sterile, molecular grade, water. We incubated samples at 37°C for 15 min, prior to the addition of 100 µl of CT Conversion Reagent. We further incubated samples, prior to purification, at (95°C for 30 s, 50°C for 60 min) for 16 cycles, then at 4°C for at least 10 min (per manufacturer's protocol). Finally, we eluted samples in 23 µl molecular grade water. We amplified the bisulfite-converted DNA using 2× KAPA HiFiSeq HS Uracil+ (KAPA, cat. no. KK2801), and unique dual index primers from NEB (cat. no. E6440S) in a final volume of 50 µl. This was performed using the following PCR program: 98°C for 45 s, followed by 17 cycles of: 98°C for 15 s, 65°C for 30 s, and 72°C for 30 s, and final extension at 72°C for 60 s. We purified and dual size selected amplified libraries using AMPure XP Beads at 0.6× to 1.0× bead ratio, eluted in a volume of 20 µl.