Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
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. Total RNA was prepared from sorted or dissociated cells with an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. For the mesendoderm-derived outgroup samples, the input to the RNA extraction kit was a cell population directly after dissociation with Accutase; for FACS sorted populations, the input was sorted cells suspended in PBS. RNA integrity was quantified with a TapeStation 4200 (Agilent). All RINe scores were ≥ 9.9. Libraries were constructed from total RNA using a Kapa mRNA HyperPrep Kit (Roche) by the Bauer Core at Harvard University.