GSM5145863: MNB_2; Drosophila melanogaster; RNA-Seq
For each scRNA-seq experiment, 120 third instar larvae of the genotype E(spl)mγGFP ;  SoxNGal4 UAS-RedStinger /TM6B were washed with PBS twice, and with 70% ethanal for 1 min, and with PBS once again. Each of the brains was dissected on ice in complete Schneider's culture medium (Schneider's Insect medium, supplemented with 10% fetal bovine serum, 2% Pen/Strep and 0.02 mg/mL insulin), and was directly transferred into a glass dish on ice containing DPBS. The dissection was completed within one hour, and then the supernatant (mainly DPBS) was replaced by 1 mL TrypLE with 1mg/mL collagenase I and 1mg/mL papain. The brains were incubated for 10 min at 30°C, with gentle shaking at 55 rpm. After removal of the dissociation solution, the brains were carefully washed with complete Schneider's culture medium once and with DPBS twice. The brains were disrupted in 1.4 ml of DPBS with 0.04% BSA by manual pipetting using a P1000, and then 0.4 ml of DPBS with 0.04% BSA was added to make a total volume of 1.8 mL. The cell suspension was filtered through the cell strainer cap into a 5mL BDFalcon FACS tube. FACS sorting was done immediately after on BD FACS ARIA II with gentle settings (85 μm nozzle and low pressure of 20 psi). Trypan blue was added before sorting. Among the singlet live cells, GFP and RFP double positive cells were selected, and sorted into DPBS with 0.04% BSA. FACS sorted cells were immediately concentrated by centrifugation at 500g for 5 minutes, then an additional 800g for 5 minutes to a 40ul volume. This entire volume was used as input for the 10x library. The single cell suspension was converted into an individually barcoded cDNA library with the Chromium Next GEM Single-Cell 3' single-index kit version 3 from 10X Genomics (Pleasanton, CA) following the manufacturer's protocols. Following ds-cDNA synthesis, sequencing library compatible with the Illumina chemistry was constructed. The final library was quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics, Ames, IA).  The final library was diluted to 5nM concentration and further quantitated by qPCR on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The final library was sequenced on one lane of an Illumina NovaSeq 6000 S1 flowcell (exp1) or a half lane of an Illumina NovaSeq 6000 S4 flowcell, as paired-reads with 28 cycles for read 1, 8 cycles for the index read, and 150 cycles for read 2.