GSM3832979: Sample 2_ovary; Drosophila melanogaster; RNA-Seq
15 – 50 LL3 ovaries were dissected per sample in ice-cold DPBS; majority of associated fat body was removed with forceps and dissection needles. For dissociation, ovaries were transferred to 9-well glass plates and incubated in dissociation solution (0.5% Type I Collagenase, 1% Trypsin 1:250 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 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 custom-made 40micron cell strainer. The strainer was built by securing nylon mesh in a cap of a 0.2 ml PCR tube and cutting the bottom of the tube and the cap. Upon filtering, dissociated cells were purified by fluorescence activated cell sorting (FACS) using a 100- micron nozzle on Sony SY3200 Cell Sorter. Single Cell 3' Reagent Kits v2: Chromium™ Single Cell 3' Library & Gel Bead Kit v2, PN-120237; Single Cell 3' Chip Kit v2 PN-120236 and i7 Multiplex Kit PN-120262" (10x Genomics)
GEO Accession
GSM3832979