GSM8879209: Purkinje_NT_H3K27ac_CUT&RUN; Mus musculus; OTHER
For CUT&RUN, Purkinje or Golgi nuclei were washed a total of two times with wash buffer (20 mM HEPES–KOH (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, Roche complete Protease Inhibitor tablet EDTA-free) and bound to activated Concanavalin A beads (Polysciences) for 10 min at room temperature. Cell permeabilization was done in 200 μl of digitonin buffer (0.05% digitonin and 0.1% BSA in wash buffer) followed by incubation with 1 μg of the antibody against H3K27ac (Abcam, ab4729) overnight at 4°C. After antibody incubation, cells were washed with digitonin buffer, followed by incubation with purified hybrid protein A-protein G-micrococcal nuclease (pAG-MNase) at 4°C for 1 hour. Samples were washed again in digitonin buffer, resuspended in 150 μl digitonin buffer, and equilibrated to 0°C on ice water for 5 minutes. To start with MNase cleavage, 3 μl of 100 mM CaCl2 was added to cells, and after 1 hour of digestion, reactions were stopped with the addition of 150 μl 2× stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 μg/ml RNase A, and 50 μg/ml glycogen). Samples were incubated at 37°C for 10 minutes to release DNA fragments and centrifuged at 16,000g for 5 minutes. Supernatants were collected, and a mix of 1.5 μl (20%) SDS/2.25 μl (20 mg/ml) proteinase K was added to each sample, followed by incubation at 65°C for 35 minutes. DNA was precipitated with ethanol and sodium acetate, pelleted by centrifugation at 4°C, washed, air-dried, and resuspended in 18 μl 0.1× TE. For CUT&RUN, the library was prepared by using the standard ChIP-seq protocol.