Single-Cell Dissociation of Embryos

Natural and ETX embryos were transferred to Falcon tubes, washed with PBS and incubated in TrypLE Express (12604013; Gibco) for 15 min at 37 °C to dissociate them into single cells. If clumps remained, the incubation was extended for an additional 5 min at 37 °C and the sample pipetted further. Samples were filtered to remove large clumps, centrifuged at 200g for 5 min and resuspended in PBST (containing 0.02% Tween 20) and then processed for encapsulation, as previously reported^{29 (link),53 (link)}. For E5.5 embryos, one litter of 12 embryos was dissociated together. A total of 15 ETX embryos were dissociated for sequencing. Cells in culture were dissociated into a single-cell suspension using TrypLE Express (12604013; Gibco) and multiplexed using MULTI-seq lipid-modified oligos before running on two 10X Genomics lanes using single-cell 3′ version 3 reagents as reported^{54 (link)}.