GSM8138704: CNR_Rest_H3k27ac_Rep2; Homo sapiens; OTHER
For ChIPseq, cells were fixed with 1% formaldehyde for 5 minutes, after quencing and cell lysis, chromatin was sonicated using a bioruptor pico (Diagenode). Sonicated samples were incubated with 5-10ug antibody for overnight and then incubated with Dynabeads Protein G for 1 hour and washed twice with sonication buffer ((10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine), once with sonication buffer with 500 mM NaCl, once with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl and 1% NP-40) and once with TE with 50 mM NaCl. After washing, DNA was eluted in freshly prepared direct elution buffer (10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 300 mM NaCl and 0.5% SDS). Cross-links were reversed by overnight incubation at 65 °C. RNA and protein were digested using RNase A and proteinase K, respectively. For CUT&RUN, cells were centrifuged for 3 min at 600 g at room temperature and then resuspended in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche Complete Protease Inhibitor EDTA-free tablet per 50 mL). Up to 500,000 cells per condition were centrifuged again for 3 min at 600 g at room temperature and resuspended in wash buffer. Activated Concanavalin-A beads (Bangs Laboratories BP531) in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) were added to each condition and incubated for 10 min on a rotator at room temperature. Beads with nuclei were kept on a magnetic stand and washed with Dig-wash buffer (0.05% digitonin in wash buffer). After discarding the liquid, beads were resuspended with primary antibody in antibody buffer (2 mM EDTA in Dig-wash buffer) and incubated overnight at 4 °C on a nutator. Then beads were washed with freshly prepared Dig-wash buffer and incubated with 1 mg/ml pAG-MNase (CUTANA) in Dig-wash buffer for 1 h at 4 °C on a nutator. After two washes with Dig-wash buffer, ice-cold incubation buffer (2 mM CaCl2 in Dig-wash buffer) was added to the beads which were subsequently placed in a metal block in an ice-water bath maintained at 0 °C for 30 min. Freshly prepared 2x Stop buffer (340 mM NaCl, 20 mM EGTA, 4 mM EGTA, 0.05% digitonin, 100 µg/mL RNase A, 50 µg/mL glycogen) was then added and incubated for 10 min at 37 °C. Beads were placed on a magnet stand and supernatant was collected. 2 µL of 10% (wt/vol) SDS and 1.5 µL of 20 mg/ml Proteinase K were added to the supernatant and incubated for 30 min at 50 °C. DNA from both ChIP and CUT&RUN samples were purified using UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (Invitrogen) following the manufacturer's instructions. Library fragments were amplified using 1× NEB next PCR master mix and 1.25 M of custom Nextera PCR primers as previously described, sing the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. The libraries were purified using a Qiagen PCR cleanup kit yielding a final library concentration of ∼30 nM in 20 µl.