GSM8223715: mESC, naive pluripotency, WT, CUT&RUN, rep1; Mus musculus; OTHER
CUT&RUN was performed as described in Skene et al. 2018 (Nature Protocols). Live cells were harvested with accutase and washed once with PBS prior to starting the procedure. Three replicates containing 200,000 mESCs or EpiLCs each were used per condition plus 2,000 HEK293T cells as a spike-in control. H3K9me2 antibody (abcam 1220) was used at 1:100 dilution in antibody buffer. Primary antibody incubation was performed at 4°C overnight. Rabbit anti-mouse secondary antibody (abcam ab6709) was used at 1:100 dilution and incubated for 4°C for 1 hour. pA-MNase (batch #6 143µg/mL) gifted from the Hennikoff lab was used for the cleavage reaction. DNA was isolated by phenol-chloroform extraction. Purified DNA was resuspended with 40µL of 1mM Trish-HCl pH 8.0 and 0.1mM EDTA. Libraries were prepared using the NEBNext Ultra II DNA Library Prep with Sample Purification Beads (NEB E7103S) following the manufacturer's protocol with 5ng of starting DNA input. Libraries were amplified using 8 cycles on the thermocycler. Each library was uniquely indexed with NEBNext Multiplex Oligos for Illumina (NEB E6440S), then pooled together in equimolar amounts. Library size was analyzed with the Agilent Bioanalyzer 2100 using the High sensitivity DNA kit.