title: GSM8838676: IMR-90, Sen (to compare with Sen-sip300/CBP), H3K27Ac CUT&RUN, rep1; Homo sapiens; OTHER
library_construction_protocol: Cells were used for CUT&RUN following the manufacturer's instructions (Cell Signaling Technologies). Cells were pelleted and suspended in Wash Buffer containing spermidine and protease inhibitor cocktail. Concanavalin A-magnetic beads were then added, and the mixture was incubated for 10 min at room temperature. Cell-bead conjugates were resuspended in 200 μl of Antibody Binding Buffer containing spermidine, a protease inhibitor cocktail, and digitonin and incubated overnight at 4℃, along with the following primary antibodies: 2 μg of anti-FOXF1 (Abcam, ab168383), 2 μg of anti-H3K27Ac (Abcam, ab4729), and 2 μg of anti-phospho-c-JUN (CST, 3270). After that, cell-bead conjugates were incubated with Digitonin Buffer containing the pAG-MNase enzyme for 1 h, followed by washing with Digitonin Buffer. Then, Digitonin Buffer containing CaCl2 was added and incubated for 30 min, followed by the addition of 150 μl of Stop Buffer containing 5 ng of S. cerevisiae spike-in DNA. After de-crosslinking, the DNA was purified using DNA Purification Buffers and Spin Columns (Cell Signaling Technologies). CUT&RUN library was prepared using a NEBNext Ultra II DNA Library Prep Kit according to the manufacturer's protocol. The anneal/extension step of PCR amplification was performed for 13 sec at 65℃ to favor exponential amplification of the desired CUT&RUN fragments. The libraries were subjected to 35-bp paired-end sequencing using the Illumina NextSeq 500/550 system (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM8838676_r1