Single cell sequencing preparation in adult Drosophila Brain

Dissect 20 male and 20 female brains from 10-day-old flies on ice-cold Schneider’s medium with FBS (Gibco, filtered 10% FBS)

After a quick spin down, remove the supernatant and wash the brains with ice-cold PBS

Incubate the brain at 25°C with 300 µl of 0.05% trypsin-EDTA (Fisher Scientific) with continuous pipetting every 5 minutes

Pass solution containing brain chunks through a 25-gauge needle (25G 5/8), without introducing air bubbles, roughly 50 times

After dissociation, the solution was through a 10 µm pluri-select cell strainer (Fisher Scientific), and 400 µl of ice-cold Schneider’s medium containing FBS was added to inactivate trypsin

Centrifuge for 0h 15m 0s at 600xg and remove the  supernatant without disturbing the pellet

Suspend pellet in sterile PBS containing 0.04% BSA

Quantify the number of cells and measure viability using an AO-PI reagent (Logos Biosystems)

Encapsulate cells, prepare and sequence libraries using the manufacturer's protocol (10X genomics & Illumina Inc.)