CUT&RUN of human cell line treated with without CD40 stimulation
Cells were collected and washed by PBS. After final wash, nuclei were isolated using nuclei extraction buffer and then washed twice with wash buffer . Five-hundred thousand nuclei were counted and incubated with activated Concanavalin A coated beads (EpiCypher 21-1411) and subsequently mixed with antibody in antibody buffer  for overnight incubation on nutator in a cold room. IgG controls were set in parrel for antibody enrichment and specificity validation. Nuclei were then washed three times with digitonin buffer and incubated with PAG-MNase (EpiCypher 15-1116) for 10 minutes at room temperature. After 10 minutes of incubation, nuclei were washed three times again with digitonin buffer and resuspended with prechilled low salt high Ca2+ buffer to initiate 1-hour digestion at 0_C (ice block). The reaction was terminated by switching into stop buffer  and incubating at 37_C for 10 minutes to release digested DNA from the nuclei. DNA was then purified with Ampure beads (Beckman A63882) size selection (0.5X to remove large fragment and 2X to recover desired nucleosomal DNA fragments). NGS libraries were generated with KAPA Hyper Prep Kits (Roche KK8502) using 14 cycles of PCR amplification. Libraires were validated on a Tapestation 4200 (Agilent G2991BA), quantified by Qubit High Sensitivity dsDNA Kit (Life Technologies Q32854), multiplexed and sequenced on a NovaSeq6000 using 100-10-10-100bp reads format.