GSM8828131: ovarian cells, Ltop4.4, replicate 1; Drosophila melanogaster; RNA-Seq
Ovaries were dissected within 30 min in ice cold Schneider's Insect Medium with 10% FBS and 167 mg/ml insulin on an ice pack. We enriched for the younger, non-vitellogenic stages of the ovary using micro-scissors. Tissue was transferred to an eppendorf tube containing ice-cold Cell Dissociation Buffer (Thermo Fisher Scientific #13151014) and rinsed once with the buffer. Dissociation was performed at RT in Cell Dissociation Buffer with 4 mg/ml elastase (Worthington Biochemical LS002292) and 2.5 mg/ml collagenase (Invitrogen # 17018-029) with nutation and regular pipetting with a P200 to aid tissue dissociation. After 20 min the solution was passed through a 50 μm filter  and the solution incubated for additional 10 min before passage through a 30 μm filter. Enzymes were quenched by adding 500 μl of dissection solution and cells were centrifuged for 5 min at 4 °C and 3500 rcf. Cells were washed in dissection solution and centrifuged again before being resuspended in ice-cold 200 μl PBS with 0.04% ultrapure BSA (Thermo Fisher Scientific AM2616). Dissociation was verified and cells were counted using a cell counting chamber and the solution adjusted to 1000 cells per μl before subjection to single-cell RNA-sequencing. The library was constructed using the Chromium Single-Cell 3' Reagent Version 2 Kit (10× Genomics) according to manufacturer's instructions.