hESC-derived cultures were dissociated with Accutase (ThermoFisher) at 37°C with trituration until nearly all clumps had been dissociated. Single-cell suspensions were filtered with a 40 μm cell strainer, washed in PBS with 1% FBS, stained with 0.5–1 μg/mL DAPI and sorted on a FACSAria II SORP (Becton Dickinson) directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. A 130 μm nozzle was used with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy.
