Instrument: Illumina HiSeq 2000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Accutase-dissociated cells were sorted using a BD Aria III (BD Biosciences) using a 100 µm nozzle. Cells were gated such that the pre-competence-loss population was taken as the cells with the top 10-15% OCT4:RFP to SOX2:YFP ratio, and the post-competence-loss population was the bottom 10-15% OCT4:RFP to SOX2:YFP ratio. We sorted around 250,000 cells per subpopulation in a typical experiment. Populations were sorted into 1.5 mL centrifuge tubes (Eppendorf) filled with 500 µL of mTeSR supplemented with 10 µM γ-27632; by the end of the sort, ~800 µL of sheath and sorted cells had been added to each tube. After the sort had completed, we pelleted the cells in a microcentrifuge at 250 xg for 3 minutes, then resuspended in PBS. For each sorted sample, about 10% of the sorted cells were reserved for competence testing to confirm the pre-/post-competence-loss status of the sorted population. These cells were seeded back into glass-bottom 24-well plates (Ibidi) treated with Matrigel and filled with 1 mL of mTeSR supplemented with γ-27632 and allowed to recover for 3 hours. The media was then changed to mTeSR supplemented with BMP4 and Activin A for 36 hours. Cells were fixed and stained for OCT4 and SOX2. ATAC-seq was performed as previously described (Buenrostro et al., 2015). Briefly, live cells were lysed and incubated with Tn5 transposase for 30 min at 37°C. After DNA purification, samples were amplified as described in (Buenrostro et al., 2015) for the appropriate number of cycles as determined by qPCR to minimize PCR bias.