Gene expression profiles at single cell level of Kenyon cells in adult Drosophila melanogaster females

Kenyon cells are grouped into 3 classes and 7 molecularly defined subtypes. While single-cell transcriptomes for the 3 classes were available from brain-level atlases, here, we take a targeted approach to distinguish between the subtypes. To generate an adult Kenyon cell data set and annotate the seven molecularly defined cell subtypes (Aso et al., 2014), we single-cell sequenced seven genotypes that simultaneously expressed GFP in all Kenyon cells and mtdTomato in a defined Kenyon cell subtype(s). To generate such flies, the OK107-QF2>QUAS-GFP, UAS-mtdTomato line (BDSC# 66472) was crossed with each of the following subtype-specific split-GAL4 lines (Aso et al., 2014; Shih et al., 2019): γm and γd (BDSC #68265), γd (BDSC #68256), α′/β′m (BDSC #68322), α′/β′ap (BDSC #68370), α/βp (BDSC #68383), α/βc (BDSC #68255), α/βs (BDSC #68267). The brains of adult female F1 progeny with the respective OK107-QF2>QUAS-GFP, GAL4-AD∩GAL4-DBD>UAS-mtdTomato genotype were dissected, visually checked for mtdTomato fluorescence in the mushroom body, dissociated into single cells, and stained with the DNA dye DRAQ5. Specifically, brains were dissected for up to 60 minutes in Schneider’s medium and placed on ice. Optic lobes were removed during dissection. After all dissections were completed, collagenase and dispase were added to a final concentration of 2mg/mL each along with 45 uM Actinomycin D, and samples were incubated at 26C for 1 h without agitation. Samples were washed 2-3 times with DPBS+0.04% BSA, dissociated by trituration and passed through a 20 um PluriStrainer. Cells were stained with 5 uM DRAQ5 before being subjected to flow cytometry on a FACS Aria II (New York University Genomics Core). Forward scatter area and width were used to gate single cells, and cells were sorted into DPBS+0.04% BSA. Specifically, DRAQ5+,GFP+ cells were FAC-sorted to enrich the samples for Kenyon cells. mtdTomato expression was not used for sorting. Given the prior knowledge that different classes of Kenyon cells separate well during the analysis of single-cell data, the brains of flies where the subtypes of different classes were labeled (e.g. γd, α′/β′ap, and α/βs) were pooled in different combinations before the dissociation to minimize batch effects. Each brain pool corresponds to one library and includes at least 8 brains. Single-cell libraries were prepared using the 10X Chromium Next GEM Single Cell 3’ Kit v3.1.