GSM9042036: HEK293T, Dox_CBPIDR7_H3K27ac, CUTRUN, Rep2; Homo sapiens; OTHER
100,000 cells per reaction or input sample were harvested and DNA fragments were prepared using the CST CUT&RUN Assay kit according to the manufacturer's instructions. In brief, 100,000 cells per reaction were harvested, washed and bound to concanavalin A coated magnetic beads. Antibodies for each reaction were added (0.5 µg IgG: Rabbit (DA1E) IgG (CST, part of CUT&RUN assay kit); 1 µg H3K27ac: Histone H3K27ac (Active Motif)) and incubated overnight at 4oC. pAG-MNase was added to each tube and incubated for 1 hour, then activated through the addiction of 3 mM CaCl2 for 30 minutes at 4oC. Stop buffer was added, supplemented with 50 pg Spike-in DNA per sample (CST, part of CUT&RUN assay kit) and DNA fragments were eluted from the beads. DNA was then purified by phenol:chloroform extraction and ethanol precipitation and resuspended in 50 µl 1x TE buffer. CUT&RUN DNA fragments were used to prepare libraries using the NEBNext Ultra II Library prep kit as described for ChIP-seq with the following changes. Adaptors were diluted for 1:10 for non-input samples and were cleaned up without size selection. Between 10-14 cycles of PCR were used for adaptor ligation dependent upon starting DNA concentration.