GSM9081588: H2A.Z, β3-AR-stimulated BAs-rep2; Mus musculus; OTHER
Cells were fixed with 1% formaldehyde for 15 minutes at room temperature, then washed three times with 1 ml wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1X protease inhibitor cocktail). The cell pellet was resuspended in 1 ml wash buffer containing 10 μl ConA-coated magnetic bead slurry, incubated at room temperature for 5 minutes, and placed on a magnetic rack to remove the solution. Beads were then resuspended in 150 μl antibody solution (wash buffer supplemented with 0.05% digitonin, 0.6 μl of 0.5 M EDTA, and 0.5 μg of H2A.Z antibody (Abcam, ab4174) or 0.5 μg Rabbit IgG and rotated overnight at 4°C. The following day, beads were washed twice with Dig-wash buffer (wash buffer supplemented with 0.05% digitonin), resuspended in 150 μl Protein A/G-MNase (700 ng/μl), and rotated at 4°C for 1 hour. After two additional washes with Dig-wash buffer, beads were resuspended in 100 μl Dig-wash buffer supplemented with 2 μl of 100 mM CaCl₂ and incubated at 0°C for 30 minutes. The reaction was stopped by adding 100 μl of 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 μg/ml RNase A, and 50 μg/ml glycogen), followed by incubation at 37°C for 30 minutes. The released genomic fragments were collected and purified. CUT&RUN  libraries were prepared by BGI.