GSM8078025: BxPC3, IgG, Replicate2, CUT&RUN; Homo sapiens; OTHER
We performed CUT&RUN in BxPC-3 cells using CUTANA ChIC/CUT&RUN Kit (EpiCypher) following the manufacturer's protocol (Skene & Henikoff, 2017). In brief, 500,000 cells were trypsinized, washed, and bound to activated Concanavalin A-conjugated paramagnetic beads (EpiCypher). The cell-bead conjugates were then resuspended in recommended buffer with 0.5 μg antibody targeting the protein of interest: Anti-BPTF (ThermoFisher Scientific A300-973A), Anti-SRF (Cell Signaling Technology 5147S), Anti-H3K4me3 (EpiCypher 13-0041k) as positive control, and Anti-IgG (EpiCypher 13-0042k) as negative control. The suspensions were incubated overnight at 4 °C with gentle shaking. Then, the cell-bead conjugates were washed twice with the kit's permeabilization buffer, and incubated with pAG-MNase (Epicypher 15-1016) for 10 min at room temperature. Subsequently, the unbound pAG-MNase was washed away, and the MNase was activated by adding CaCl2 to a final concentration of 2 mM. The reaction was stopped after the pAG-MNase was allowed to digest and release the antibody-bound chromatin for 2 hours at 4 °C. Finally, the released DNA was purified with the spin columns provided by the kit (EpiCypher). Library preparation was performed using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). The libraries were sequenced on a Illumina NextSeq 500 using 2x75bp reads.