title: GSM7698846: U937_FICZ_Replicate_1; Homo sapiens; OTHER
library_construction_protocol: Cells were washed twice with room temperature (RT) Wash Buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, Protease inhibitor cocktail (1:100)). After wash, cells were resuspended with RT Wash Buffer and incubated with activated Biomag Plus Concanavalin A (ConA) beads (BANGS LABORATORIES INC, BP531) for beads slurry. Cell-binding beads were incubated with freshly made antibody buffer (Wash Buffer, 0.01% Digitonin, 2mM EDTA) and primary antibody at 4°C overnight with rotation. The next day, beads were washed twice with ice-cold Digitonin Buffer (Wash Buffer with 0.01% Digitonin) and incubated with 2.5ul/sample CUTANA PAG-MNase (EpiCypher, 15-1116) to allow the binding of PAG-MNase.  After another 2 times washes with cold Digitonin Buffer, beads were incubated with 1ul/sample 100MM CaCl2 4°C on nutator for 2 hours to ensure efficient chromatin digestion. 33ul/sample Stop Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/ml RNase A, 50ug/ml Glycogen) was added to stop MNase activity. DNA purifying was performed using the DNA Clean & Concentrate -5 kit (Zymo Research, D4014) as per manufacturer's instructions. Briefly, 5 volumes of DNA Binding Buffer was added to each sample and washed twice with DNA Wash Buffer. Fragment DNA was eluted with 23ul/sample 0.1x TE Buffer and proceed to library preparation. NEBNext Ultra II Library Prep kit was used for library preparation. CUT&RUN libraries were sequenced on the NovaSeq PE150 platform.