Wild-type embryos from Canton S flies were laid on apple-agar plates in two-hour or four-hour windows, left to age for the specified time window, then collected from the plates, cleaned and dechorionated. Embryos were dissociated in ice-cold hypotonic lysis buffer with a Dounce homogenizer, filtered through a cell strainer, and nuclei were cross-linked on ice for 30 minutes.

Trimmed reads were mapped to the Drosophila reference genome. Further processing was performed with Seurat v3. For quality filtering, cells with fewer than 250 UMIs, cells with more than 10,000 UMIs, cells with reads mapping to more than 7,000 genes, and cells with more than 10% of read counts mapping to ribosomal genes, were excluded. Dimensionality reduction and clustering was performed using the standard Seurat processing pipeline, with a sample-dependent resolution.