GSM8759317: mESC, primed pluripotency, WT, LAP2B, rep1; Mus musculus; OTHER
CUT&RUN was performed as described in Skene et al. 2018. 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, except for H3K9me3 CUT&RUN which only included two replicates of wildtype naïve mESCs. LAP2B antibody (Invitrogen PA5-52519, 1:100), H3K9me3 antibody (abcam 8898, 1:1000 dilution), and Lamin B1 antibody (sc56144, 1:500) were diluted in antibody buffer and incubated at 4°C overnight. Rabbit anti-mouse secondary antibody (abcam ab6709, 1:100 dilution) was used for as an IGG negative control in parallel with the LAP2B CUT&RUN. pA-MNase (batch #6 143µg/mL) generously gifted from the Hennikoff lab was used for the cleavage reaction. The 2X STOP buffer included genomic DNA from Saccharomyces cerevisiae such that each sample received 0.3pg of spike-in DNA as a spike-in control. The chromatin release step was carried out at 37°C for 1 hour. DNA was isolated by phenol-chloroform extraction. Purified DNA was resuspended with 40uL 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.