Embryos were incubated on yeasted apple juice plates at 25C for 2.25h before being dechorionated in 2.6% sodium hypochlorite, rinsed with water and suspended in PBS 0.5% Truton X-100. They were then resuspended in ice-cold dissociation buffer and dissociated with a clean metal pestle. Cells and tissue fragments were pelleted and then gently resuspended in trypsin-EDTA, pelleted again, and resuspended in dissociation buffer.

Dmel R6.32

Reads were processed using Cell Raner v3.1.0 (10x Genomics). They were aligned to the Ensembl 98 Drosophila melanogaster genome. Quality control analyis was performed with scater v1.14.6. Doublets were identified and removed using scDblFinder v1.1. Further analysis was performed with Seurat v3.1.4. Clustering was performed using the shared nearest neighbor approach, using the first 12 dimensions from the principal components analysis, a k parameter value of 60 and a clustering resolution of 0.5.