GSM5618627: H3K27me3_rep1; Rattus norvegicus; OTHER
Oocytes were incubated with rabbit anti-H3K27me3 antibody, rabbit anti-H3K4me3 antibody, or rabbit anti-H3K36me3 in a wash buffer (20 mM HEPES (pH7.5), 150 mM NaCl, 0.5 mM Spermidine, complete EDTA-free protease inhibitor cocktail, 0.02% Digitonin) containing 2 mM EDTA at 4C on a plastic dish overnight. After the oocytes were washed twice by the wash buffer on a plastic dish, they were incubated with Protein A-MNase (500 ng/ml) provided by the Steven Henikoff Lab at 4C for 3 hrs on a plastic dish. After washing twice, the oocytes were transferred into a 1.5 ml tube containing pre-activated BioMag Plus Concanavalin A (ConA) beads. The sequential step of the antibody incubation followed by the ConA binding prevents loss of oocytes during wash steps. After the oocytes were bound to ConA beads and the buffer was replaced to the wash buffer, CaCl2 was added at a final concentration of 2 mM to activate the MNase. After 20 min of digestion on ice, the digestion reaction was stopped and DNA fragments were recovered. CUT&RUN libraries were prepared as previously described (Inoue et al., Genes Dev., 2018). PCR amplification was performed for 11-15 cycles.