title: GSM8001897: mESC, synHPRT1RnoCpG integrated on chrX, clone 1, PolII ChIP, rep 2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were grown to medium confluency in 6-well plates. Cells were harvested by washing once with PBS, disso-ciating into single-cell suspending with TrypLE Express (ThermoFisher 12604013) and then neutralizing with mESC media. Crosslinking was performed by adding formaldehyde to a final concentration of 0.1% (v/v), and cells were incubated at room temperature for 5 minutes with occasional mixing by in-version. Crosslinking was stopped by quenching with 125 mM glycine, incubating at room temperature for 5 minutes with occasional mixing by inversion. DMSO was added to a final concentration of 10% (v/v) and cells were frozen in aliquots of ~106 cells. ~10^6 crosslinked and frozen mESCs were thawed and processed for CUT&RUN using the CUTANA™ ChIC/CUT&RUN kit (EpiCy-pher 14-1048) according to the manufacturer's protocol. Antibodies were all used at 0.5 μg: rabbit IgG negative control (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), H3K27ac (EpiCypher 13-0045), H3K27me3 (Active Motif 39055, RRID: AB_2561020), Pol II (Santa Cruz Biotechnology sc-56767). Sequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs E7645L) according to the manufacturer's protocol.