GSM4946264: adult4; Drosophila melanogaster; RNA-Seq
For each sample, 30-40 pairs of adult ovaries were dissected one by one in ice-cold DPBS, no calcium, no magnesium (Thermo Fisher Scientific #14190136). Abdomens were removed using forceps and parts of intestine and abdominal cuticle were removed. Using forceps and a dissection needle, we cut off the anterior tip of the ovary and transferred to a new well. Method B: For dissociation, anterior tips of ovaries we transferred to a 1.5 ml tube and were incubated in dissociation solution (0.25 mg/ml Type I Collagenase, 0.4 mg/ml elastase (Worthington Biochemical #LS002290) in Cell Dissociation Buffer (Thermo Fisher Scientific #13151014)) for 30 minutes with gentle rotation. The suspension was vigorously pipetted every 5 minutes during the dissociation to enhance the dissociation efficiency. Enzymatic dissociation was stopped by adding Schneider cell culture media with fetal bovine serum (S-FBS). Starting from this step, all plastic materials – pipet tips, tubes, filters – were coated with S-FBS. Cell suspension was filtered through a 70-micron filter (Miltenyi Biotec #130-095-823), and 30 micron filter (Miltenyi Biotec #130-041-407). Cell suspension was centrifuged for 5 minutes at 3500 rcf at 40C. Cells were resuspended in 0.04% ultrapure BSA (Thermo Fisher Scientific #AM2616) in DBPS. 10x Genomics Chromimum v3 kit
GEO Accession
GSM4946264