GSM5939844: young control gut cells [YMC]; Drosophila melanogaster; RNA-Seq
Guts of anesthetised adult female flies were dissected in PBS. Proventriculus and hind gut were carefully removed. The dissected midguts were placed in ice-cold Schneider's medium (without FBS or penicillin/streptomycin). Collected midguts (100 midguts/sample) were washed briefly with PBS and transferred to a 1.5 mL eppendorf tube containing 5 mg/mL elastase/PBS solution (SIGMA, E0258). The midgut solution was incubated on a shaker at 27℃, 1,000 rpm for 25 min with pipetting (40 times) every five minutes. Pipette tips were pre-coated with the elastase solution to prevent midgut from sticking to them. The dissociation reaction was stopped by adding 400 μL of the Schneider's medium. The cell suspension was passed through a 100 μm cell strainer. The flow through was centrifuged at 600 × g for 20 minutes and the supernatant was discarded. The cell pellet was resuspended in 100 μL of the Schneider's medium. Cell number and viability were assessed by trypan blue staining using a hemocytometer. A single cell suspension was obtained by filtering with a 40 μm cell strainer. Library preparation were performed with the Single Cell 3' Library & Gel Bead Kit v3.1 according to the 10X Genomics' standard protocol.