GSM5739895: 40 day post-fertilization zebrafish ovaries cells (zx2); Danio rerio; ssRNA-seq
GSM5739895
Somatic ovary cells were dissociated using a modification of Elkouby and Mullins (2016). 40 pairs of 40 dpf ovaries were dissected from Tg(piwil1:egfp) transgenic fish (Leu et al., 2010) were dissected and stored in a LoBind tube containing 2mL of L15. Tissues were minced with microdissection scissors into < 1 mm pieces. After the tissue had settled to the bottom of the tube, the media was replaced with 5mL of digestive enzyme mixture (3 mg/mL collagenase I (Cat.No. C0130; Sigma-Aldrich), 3 mg/mL collagenase II (Cat.No. C6885; Sigma-Aldrich), and 1.6 mg/mL hyaluronidase (Cat.No. H4272; Sigma-Aldrich) in L15 and incubated on an orbital rotator at room temperature . The suspension was monitored every 10 mins until a minimal number of cell clumps were observed (~30 min.). Cells were centrifuged for 3 mins at 300 x g and resuspended in 5 mL of 5x TrypLE (Cat.No. A1217701; Thermo Fisher) in L15 and incubated on an orbital rotator at room temperature for 15 min. The trypsin reaction was stopped by adding 500 µL of 2.8 mg/mL trypsin inhibitor in L15 and incubated on an orbital rotator at room temperature for 1 min. The cell suspension was then added to 25 ml of L15 in a 50 ml conical tube to dilute the trypsin and centrifuged for 3 min at 300 x g. The cell pellet was resuspended and washed two times with 5 mL of L15 using a P1000 pipette and centrifuged for 3 mins at 300 x g. The cell pellet was then resuspended and filtered as described above. 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.