GSM8405590: H3f3a_AA_H3f3b_WTA_clone1_CUTRUN_K27ac; Mus musculus; OTHER
100,000 nuclei were isolated from cell populations using a hypotonic buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5mM spermidine, 0.1% Triton X-100, 20% glycerol, freshly added protease inhibitors) and flash frozen. Nuclei were thawed and bound to lectin-coated concanavalin A magnetic beads (50 µL bead slurry per 100,000 nuclei; Polysciences). Immobilized nuclei were pre-blocked with blocking buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5mM spermidine, 0.1% BSA, 2 mM EDTA, fresh protease inhibitors) and washed in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5mM spermidine, 0.1% BSA, fresh protease inhibitors). Nuclei were incubated in wash buffer containing primary antibody (H3.3, K122ac, K27ac) overnight at 4°C with rotation. Nuclei were incubated in wash buffer containing primary antibody for 1 hour at room temperature with rotation. Next, samples were washed twice with wash buffer and incubated in wash buffer containing recombinant pA-MN (for K122ac, K27ac) or pA/G-MN (for H3.3) for 30 minutes at room temperature with rotation. Controls lacking a primary antibody were subjected to the same conditions but incubated in wash buffer without any primary antibody prior to incubation with pA-MN/pA/G-MN. After incubation, samples were washed twice with was buffer and equilibrated to 0°C in an ice/water bath. MNase cleavage was activated upon addition of 3 mM CaCl2. Digestion was chelated for samples containing primary antibodies K27ac, after 30 mins on the ice water bath, using 20 mM EDTA, 4 mM EGTA, 200 mM NaCl, and 1.5 pg MNase-digested S. cerevisiae mononucleosomes were added as a spike-in control. Genomic fragments were released after an RNase A treatment. Released fragments were separated through centrifugation. Digestion was chelated for samples containing primary antibodies K122ac and H3.3 after 30 mins on the ice water bath, using a low-salt treatment: 10 mM EDTA, 2 mM EGTA, 150 nM NaCl, 5 mM TritonX, and 1.5 pg MNase-digested S. cerevisiae mononucleosomes were added as a spike-in control. Genomic fragments were released after being incubated for 1 hour at 4°C and released fragments were separated from beads. The salt concentration of low-salt treated samples were increased to 500 mM and additional RNase A. Isolated fragments from all samples were used as input for a library build consisting of end repair and adenylation, adapter ligation, and subsequent purification with AMPure XP beads (Agencourt). Barcoded fragments were then amplified by 15 cycles of high-fidelity PCR and purified using agarose gel extraction. Libraries were pooled and sequenced on an Illumina NextSeq500 to a depth of ~10 million mapped reads.