Drosophila pupae of the line SS000324-Gal4 recombined with UAS- mCD8::GFP were collected at tehe desired development stge and dissected in Schneider's insect medium. Brain cells were dissociated with a mix of papain and Liberase in complete Schneider's medium for 30 minutes at 30C. GFP-positive cell bodies were isolated from the sample using a BD Aria III cell sorter.

Reads were processed using Cell Ranger v3 (10x Genomics). They were aligned to the Ensembl 97 Drosophila melanogaster genome. Further analysis was performed using Seurat v3.1.0.9007. For quality filtering, genes expressed in less than 3 cells and cells with less than 200 UMIs, as well as cells with more than 10% of counts allocated to genes coding for mitochondrial or heatshock proteins, were excluded. Clustering was performed using the first 15 principal components and a resolution parameter of 0.8 with the Louvain algorithm.