GSM5133563: WT-PBS-1_H3K27ac; Mus musculus; ChIP-Seq
Samples containing 1M cells were centrifuged at 1600 g for 5 minutes at 4°C. Each sample was washed twice with cold 1 mL PBS and resuspended in 9.375mL of PBS. Then, 0.625 mL of 16% formaldehyde was added and the samples were incubated on a shaker for 5 minutes at room temperature. The samples were then quenched with 0.667 mL of 2M glycine and incubated for 5 minutes at room temperature on a rotator. The cells were then centrifuged at 1600 g for 5 minutes and washed twice with 1 mL cold PBS. The pellet was resuspended in 130 µL of Covaris sonication buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1% SDS and 1x protease inhibitor cocktail (PIC)) and sonicated with a Covaris S220 sonicator using 75 W peak incident power, 5% duty factor, and 200 cycles per burst for 16 minutes at 4°C. Sonicated samples were centrifuged at 16,100 g for 10 minutes at 4°C before the supernatant containing sheared chromatin was removed to a fresh tube. 2.4 µL of sheared chromatin was then mixed with 46.6 µL of IP buffer (20mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100, 1% freshly added PMSF and PIC) to generate a 50µL sample containing chromatin from 20,000 cells for MOWChIP-seq. Libraries for sequencing were prepared using the Accel-NGS 2S Plus DNA Library Kit (Swift-Bio).