GSM5739894: 40 day post-fertilization zebrafish ovaries cells, sorted germ cells; Danio rerio; ssRNA-seq
GSM5739894
Germ cells were dissociated from the somatic gonad using a modification of Blokhina et al., (2019). 40 pairs of 40 dpf ovaries were dissected from Tg(piwil1:egfp)uc02 transgenic fish (Leu et al., 2010) and stored in a LoBind tube (Cat.No. 0030108302; Eppendorf) containing 2 mL of L15 medium(Cat.No. L5520; Sigma-Aldrich). The tissue was minced with small scissors into <1 mm pieces. 200 µL of 20 mg/mL of type 2 collagenase in L15  (Cat.No. NC9870009; Worthington) were added and incubated on an orbital rotator at 28°C for 35 min. The cell suspension was then gently passed through a 23g needle five times to break up large cell clumps. 200 µL of 7 mg/mL trypsin (Cat.No. LS003708; Worthington) in L15 were added and incubated on an orbital rotator for 10 min or until a minimal amount of cell clumps was observed. The trypsin reaction was stopped by adding 500 µL of 20 mg/mL trypsin Inhibitor (Cat.No. 100612; MP Biomedicals) in L15. The cells were centrifuged for 3 min at 300 x g and the supernatant carefully removed. The cells were then resuspended and washed two times with 5 mL of L15 using  a P1000 pipette tip and then centrifuged for 3 min at 300 x g. Cells were then resuspended in 1 mL L15 and filtered through a 100 µm nylon filter (Cat.No. 431752; Corning) and then through a 40 µm nylon filter (Cat.No. 431750; Corning) to remove cell clumps. The filtrate was centrifuged for 3 min at 300 x g and resuspended in 1 mL of 50 mg/mL BSA (Cat.No. A8806; Sigma-Aldrich) in Phosphate Buffered Saline. Cell viability and number were assessed using propidium iodine (Cat. No. P1304MP; Thermo Fisher) and Hoechst 33342 (Cat.No. H3570; Thermo Fisher) staining on a Fuchs-Rosenthal hemocytometer (Cat.No. DHC-F01; Incyto). Following cell dissociation of Tg(piwil1:egfp) transgenic ovaries, GFP+ germ cells were sorted using a MoFlo Astrios EQ Cell Sorter (Beckman Coulter) with a 70 µm nozzle. Cells were sorted using side scatter and GFP purify to identify single germ cells. Single-cell RNA sequencing libraries were prepared by the UC Davis DNA Technologies core. Briefly, barcoded 3' single-cell libraries were prepared from dissociated cell suspensions or sorted cells using the Chromium Single-Cell 3' Library and Gel Bead kit V3 (10X Genomics) for sequencing according to the manufacturer's recommendations. All libraries were targeted at 10,000 cell recovery and were amplified using 11 cycles. The cDNA and library fragment size distribution was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent).  The libraries were quantified by fluorometry on a Qubit instrument (LifeTechnologies) and by qPCR with a Kapa Library Quant kit (Kapa Biosystems) prior to sequencing. Libraries were sequenced on a HiSeq 4000 sequencer (Illumina) with paired-end 100 bp reads.