title: GSM6737991: typeII lineage, RFP+, scRNA-seq; Drosophila melanogaster; RNA-Seq
library_construction_protocol: UAS-dcr2; wor-gal4, ase-gal80; UAS-RFP::stinger larval brains (n=50) were dissected 96hr after larval hatching in ice cold Rinaldini's solution during a 45-min interval. Dissected brains were transferred to Eppendorf tubes containing 30µL of Rinaldini's solution. A total of 10µL of 20mg/mL papain, 10uL of 20mg/mL type-1 collagenase, and 1µL of 15µM ZnCl was added to the tube. Additional Rinaldini's solution was added to adjust the final volume to be 100 µL. The tube was mixed gently by flicking, and them incubated on a heat block at 37°C for 1-hr, while covered with aluminum foil. During this incubation, the tube was flicked for mixing every 10 min.     After the 1-hr incubation, 5µL of 100µM E-64 was added to stop the papain digestion. Samples were incubated on ice for 2 min, and then centrifuged for 3 min at 500g. Supernatant was carefully removed, and chemical dissociated brains were resuspended in 100µL Schneider's media with 10% fetal bovine serum (FBS). Mechanical dissociation was performed by setting a P100 pipette to 70µL and titrating 30 times at a frequency of ~1Hz. After titration, cells were diluted with 400µL Schneider's media with 10% FBS, bringing the total volume to 500µL. A total of 1µL of DRAQ5 DNA stain (ThermoFisher Scientific) was added to label cells apart from debris.     A Sony MA900 FACS machine was used to select for RFP+, DRAQ5+ cells. Cells were sorted into a 1.5mL Eppendorf tube prefilled with 100µL of Schneider's media with 10% FBS. Approximately 30,000 RFP+, DRAQ5 events were sorted. . Cells were transported on ice to the University of Michigan's Advanced Genomics Core and were loaded for 10X Chromium V3 sequencing following the manufacturer's instructions.      The mRNA was subsequently reverse-transcribed, amplified, and sequenced on an IlluminaNovaSeq-6000 chip (University of Michigan Advanced Genomics Core). Then, 151-bp paired-end sequencing was performed, with a target of 100,000 reads/cell.