scRNAseq of Drosophila melanogaster: VNC at pupal stages
L3 stage larvae were collected with forceps, sexed under a dissecting scope and put in either male or female vials to develop until the correct stage, i.e. 6, 24, 36 or 48 hours after puparium formation (h). Ventral nerve cords (VNCs) were dissected in ice-cold DPBS (Dulbeccos Phosphate Buffer Saline, Gibco). A maximum of 8 VNCs, each of a different genotype, were pooled in a low-bind eppendorf tube containing 100l of DPBS on ice. VNCs from animals of two or three different developmental stages and sex were pulled in the same tube (6+24h, 24+48h or 24+36+48h stages). After dissections, tissues were collected at the bottom of the tube by centrifugation (800g, 5 mins, 4C), DPBS was replaced with 50l of dispase I (3mg/mL, Sigma-Aldrich, Cat. D4818, reconstituted in 50mM HEPES/KOH pH 7.4, 150mM NaCl) and 75l of collagenase I (100mg/mL, Invitrogen, Cat. 17100-017, reconstituted in HBSS). VNCs were dissociated in a thermo mixer (Eppendorf) at 25C at 500rpm for 30 (samples with 6+24h stages) or 40 mins (samples with 24+36+48h stages). Dissociation was aided by pipetting up and down 10 times every 10 mins ensuring that the tip touched the bottom of the tube to increase the mechanical stress. Dissociated cells were centrifuged (800g, 5 mins, 4C), washed once with DPBS and resuspended in 180l of DPBS with 0.04% BSA. Cell suspension was filtered through a 10m filter (pluriStrainer, Cambridge Bioscience, Cat. 43-50010-03). Viability, presence of debris and cell number were accessed by a Countess II automated cell counter (Invitrogen). Samples were collected on 3 different days. Each day a total of 4 different cell suspensions (independent samples) were generated, each containing different combinations of genotypes, ages and sex. Single cell expression libraries were generated at the CRUK-CI sequencing facility (Cambridge, UK) using ChromiumTM Next GEM Single Cell 3 HT v3.1. We aimed to load each sample in duplicate, 20,000 cells per lane. Before sequencing, libraries were inspected on a tapestation.