title: scRNA-seq of Drosophila melanogaster: central brain
design: We first used the split-pool method (Parse Bioscience, Seattle, WA, USA) to barcode RNAs from whole brain to generate a library which includes both T1 and T2 progeny. This library, T1+T2, was used for analysis in Figure 1 and 5. To increase the number of T2 nuclei for snRNAseq, we labeled T2 nuclei by crossing 20xUAS-FLPG5.PEST;worniu-gal4,asense-gal80; Act5c(FRT.CD2)gal4  to UAS-RedStinger (RFP) or UAS-unc84-2xGFP (GFP) flies. The adult flies were aged for 1 week at 25 C before dissection. Equal amounts of male and female central brains (excluding optic lobes) were dissected at room temperature within 1 hour. The samples were flash-frozen in liquid nitrogen and stored separately at -80C. Nuclei dissociated from the frozen, dissected brains was performed according to published protocols (McLaughlin et al., 2022). RFP+ or GFP+ nuclei were collected by sorting dissociated nuclei with SONY-SH800 with 100 m chip. We performed three rounds of sorting and snRNAseq. In the first round, we pooled male and female brains together to select GFP+ nuclei and used particle-templated instant partitions to capture single nuclei to generate cDNA library (Fluent BioSciences, Waterton, MA). In the second round, RFP+ nuclei from male and female were pooled together. In the third round, RFP+ nuclei from male and female brains were collected separately. The split-pool method was then used to generate barcoded cDNA libraries from each individual nucleus from the second and third rounds. All libraries were sequenced with pair-ends reads 150bp on Illumina Novaseq 6000 (University of Oregon'sGenomics and Cell Characterization Core Facility).