GSM4946261: adult1; 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 A: For dissociation, anterior tips of ovaries were incubated in dissociation solution (0.5% Type I Collagenase (Thermo Fisher Scientific #17018029), 1% Trypsin 1:250 (Thermo Fisher Scientific #27250018) in DPBS) for 15 minutes with gentle rotation. The suspension was vigorously pipetted multiple times during the dissociation to enhance the dissociation efficiency. Enzymatic dissociation was stopped by adding Schneider cell culture media (Fisher # 21720-001) with 10% fetal bovine serum (Invitrogen #10082-147) (S-FBS). Starting from this step, all plastic materials – pipet tips, tubes, filters – were coated with S-FBS. Cell suspension was filtered through a custom-made ~40-micron cell strainer. The strainer was built by securing nylon mesh (Component Supply #U-CMN-40) in a cap of a 0.2 ml PCR tube and cutting the bottom of the tube and the cap (Slaidina et al. 2020). Upon filtering, dissociated cells were purified by fluorescence activated cell sorting (FACS) using a 100-micron nozzle on Sony SY3200 Cell Sorter. Cell suspension was centrifuged for 5 minutes at 250 rcf at RT, and resuspended in S-FBS. 10x Genomics Chromimum v2 kit
GEO Accession
GSM4946261