GSM6046071: H3K27Ac_PPO2_1; Mus musculus; OTHER
GSM6046071
CUT&RUN was performed following the published protocol (Skene and Henikoff, 2017). 200,000 cells were harvested by centrifugation (600 g, 3 min) and. Cells were washed by in 1 ml Wash Buffer (20 mM HEPES-KOH pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1x Roche cOmpleteTM) for twice followed by addition of 10 µl pre-activated concanavalin A coated magnetic beads (Bangs Laboratories-BP531). Tubes were held on mixing platform at RT for 10 mins, then placed on a magnet stand and pull off the liquid. Add 100 µL cold Antibody Buffer to each sample, and 1 µL antibody (1:100 dilution) to each sample and incubate on nutator overnight at 4ºC. Beads with cells were washed in 1 ml Digitonin Buffer (20 mM HEPES-KOH pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1x Roche cOmpleteTM,0.05% digitonin.) for twice, and resuspended in 50 µL Digitonin Buffer. Add 2.5 µL CUTANA pAG-MNase (20x stock) to each sample, and gently vortex. Incubate samples for 10 min at RT. Beads with cells were washed in 1 ml Digitonin Buffer for twice. Remove liquid and add 50 µL cold Digitonin Buffer to each sample, keep sample in 0 ºC block. Add 1 µL 100 mM CaCl2 to samples, incubate for 30 min in 0 ºC block. Add 50 µL Stop Buffer to each sample, and gently vortex to mix. Incubate tube for 30 min at 37ºC for release cleaved fragments. Cleaved fragments were purified by Qiagen MinElute PCR Purification Kit.  DNA fragments were extracted from the supernatant and used for the construction of sequencing libraries. Libraries were prepared for Illumina sequencing with Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645).  Libraries were sequenced by NovaSeq PE150