STAT1_rep3_Naive0h
Homo sapiens
Protocols: HeLa cells which primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. samples were processed using CUTANA™ CUT&RUN kit (EpiCypher) according to the manufacturer's instructions, cells were fixed with 1% formaldehyde for 1 minute at room temperature, followed by quenching in 25mL of 125mM glycine in PBS. Cells were harvested by low-speed centrifugation, 500,000 cells were washed with washing buffer, then incubated with pre-activated Concanavalin A-coated beads for 10 minutes at room temperature, followed by an overnight incubation at 4°C with antibodies (0.5μg) in antibody-buffer containing 0.01% digitonin. Next, ConA beads bound cells were washed twice with permeabilization buffer containing 0.01% digitonin, then incubated with protein A-MNase fusion for 10 minutes at room temperature and washed to remove unbound protein A-MNase. Cleavage was performed at 4°C for 2h by addition of calcium chloride to a final concentration of 100mM. After incubation, reaction was stopped by addition of the STOP buffer (containing fragmented genomic E. coli DNA as spike-in). All cell lines were incubated at 37oC, 5% CO2, and grown in 'Dulbecco's Modified Eagle Medium (DMEM) containing high glucose and pyruvate (ThermoFisher, 41966-029) supplemented with 10% NCS (newborn calf serum, ThermoFisher, 16010-159) and 1% Penicillin-Streptomycin (ThermoFisher, 15140-122). Fragmented DNA samples were extracted after 10 minutes of incubation at 37°C, followed by phenol-chloroform extraction. For preparation of DNA libraries, fragmented DNA was processed using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions.