title: scRNA-seq of Drosophila melanogaster
design: Single cell sample preparation: Male flies of the wg-Gal4 line were crossed to virgin females from UAS-Red Stinger (BL#8547) to label nuclei of specific wg-Gal4-positive cell types. Brains were dissected from wg-Gal4 wandering third instar larvae in ice-cold Schneiders medium (Gibco), complemented with 10% Fetal Bovine Serum, 1% Penicillin-Streptomycin, 200mM L-Glutamin, 4mg/ml L-Glutathione and 20ug/ml insulin (all from Sigma-Aldrich). Dissected brains were dissociated into a single cell suspension by incubating in 2mg/ml collagenase and 2mg/ml papain (Sigma-Aldrich) in complemented Schneiders medium for 30 min at 25C. The enzymes were then removed and replaced with complemented Schneiders medium. The brains were further mechanically dissociated by gently pipetting up and down the content of the tube 300 times until most large chunks of tissue are dissociated. The sample was then filtered using 20-m cell strainers (pluriSelect) and kept cold by putting tube on ice. The cells expressing the transgene were sorted on the basis of their fluorescent signal using Fluorescence-Activated Cell Sorting (FACS Aria III). The cells were sorted in a tube containing PBS + 0.1%BSA. The concentration of cells was calculated based on FACS parameters. Library preparation, sequencing, and processing: Towards the end of sorting, we placed all appropriate reagents (as indicated in the manufacturers protocol, Chromium Single Cell 3 Reagent Kits v3 chemistry) to equilibrate at room temperature. Droplet-based purification, amplification and barcoding of single-cell transcriptomes were performed using Chromium Single Cell 3 Reagent Kit v3 (10x Genomics) as described in the manufacturers manual, with a target recovery of 8,000 cells per experiment. We prepared 15 libraries which were subjected to paired-end sequencing (26  8  98) using the Illumina NovaSeq 6000 system (New York University Abu Dhabi) to an average 50,000 reads per cell sequenced. We mapped the sequenced libraries to the Drosophila melanogaster genome assembly BDGP6.88 using CellRanger 3.0.1.