{"database": "metadata", "table": "run_metadata", "rows": [[67760, "SRR17262952", "SRX13441048", "SRS11340477", "SRP351377", "PRJNA789855", "Single cell transcriptome reveals insights into the development and function of the zebrafish ovary", "GSE191137", "Transcriptome Analysis", "Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development  yet there are still significant gaps in our understanding of the genes that regulate gonad development  sex  and reproduction. Unlike the development of many organs  such as the brain and heart that form during the first few days of development  zebrafish gonads do not begin to form until the larval stage  =5 dpf. Thus  forward genetic screens have identified very few genes required for gonad development. In addition  bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations  we have used single cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10 658 germ cells and 14 431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types  including follicle cells  theca cells and interstitial stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance  we determined the location of these cell subpopulations within the ovary. Finally  for select examples  we used gene knockout experiments to determine the role of newly identified genes. Our results reveal novel insights into ovarian development and function and the sequencing information will provide a valuable resource for future studies. Overall design: Single cell RNA sequencing of 40 dpf zebrafish ovaries", null, "pubmed:35588359", null, "40 dpf zebrafish ovaries cells  sorted germ cells", "GSM5739894", null, "tissue:40 dpf zebrafish ovaries|cell type:Germ cells|genotype:AB wildtype", "40 dpf zebrafish ovaries cells  sorted germ cells", "A General Transfer Format GTF gene annotation filerelease 96 for the GRCz11 zebrafish genome was downloaded from Ensembl Genome Browser and filtered using the \u201cmkgtf\u201d function in Cell Ranger v3.0.2; 10x Genomic to retain the following attributes: protein coding  lincRNA  and antisense. A genome reference file was generated with Cell Ranger\u2019s \u201cmkref\u201d function using the GRCz11 zebrafish genome obtained from the Ensembl Genome Browser with alternative loci scaffolds removed and the filtered GTF file. GRCz11 zebrafish genome FASTQ file from Ensembl Genome Browser with alternative loci scaffolds removed and the filtered GTF file described above were used to generate the count file using the \u201ccount\u201d function in Cell Ranger v3.0.2; 10x Genomic. \u201cexpect cells\u201d was setted to 10000 based on estimated cell recovery.\u00a0 Genome build: GRCz11 Supplementary files format and content: Filtered or raw matrix files generated from Cell Ranger in one zip file.", "40 dpf zebrafish ovaries", null, "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 Tgpiwil1:egfpuc02 transgenic fish Leu et al.  2010 and stored in a LoBind tube Cat.No. 0030108302; Eppendorf containing 2 mL of L15 mediumCat.No. L5520; Sigma Aldrich. The tissue was minced with small scissors into <1 mm pieces. 200 \u00b5L of 20 mg/mL of type 2 collagenase in L15  Cat.No. NC9870009; Worthington were added and incubated on an orbital rotator at 28\u00b0C for 35 min. The cell suspension was then gently passed through a 23g needle five times to break up large cell clumps. 200 \u00b5L 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 \u00b5L 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 \u00b5m nylon filter Cat.No. 431752; Corning and then through a 40 \u00b5m 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 Tgpiwil1:egfp transgenic ovaries  GFP+ germ cells were sorted using a MoFlo Astrios EQ Cell Sorter Beckman Coulter with a 70 \u00b5m 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\u2019 single cell libraries were prepared from dissociated cell suspensions or sorted cells using the Chromium Single Cell 3\u2019 Library and Gel Bead kit V3 10X Genomics for sequencing according to the manufacturer\u2019s 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.", "Zebrafish husbandry was performed as previously described Westerfield  2000.", "cell type:Germ cells|genotype:AB wildtype", "GSM5739894", "GSM5739894: 40 dpf zebrafish ovaries cells  sorted germ cells; Danio rerio; ssRNA seq", "GSM5739894 r1", "GSM5739894", "1", "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 Tgpiwil1:egfpuc02 transgenic fish Leu et al.  2010 and stored in a LoBind tube Cat.No. 0030108302; Eppendorf containing 2 mL of L15 mediumCat.No. L5520; Sigma Aldrich. The tissue was minced with small scissors into <1 mm pieces. 200 \u00b5L of 20 mg/mL of type 2 collagenase in L15  Cat.No. NC9870009; Worthington were added and incubated on an orbital rotator at 28\u00b0C for 35 min. The cell suspension was then gently passed through a 23g needle five times to break up large cell clumps. 200 \u00b5L 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 \u00b5L 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 \u00b5m nylon filter Cat.No. 431752; Corning and then through a 40 \u00b5m 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 Tgpiwil1:egfp transgenic ovaries  GFP+ germ cells were sorted using a MoFlo Astrios EQ Cell Sorter Beckman Coulter with a 70 \u00b5m 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 three prime single cell libraries were prepared from dissociated cell suspensions or sorted cells using the Chromium Single Cell three prime 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.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP351377", null, null, "40d_Germ_S67_L007_R1_001.fastq.gz 40d_Germ_S67_L007_R2_001.fastq.gz", "fastq fastq", 97157899800.0, 321714900.0, "GSM5739894 r1", "0:151 1:151", "A:25130180865;C:15016964022;G:17255588720;T:39751154898;N:4011295", 151, 151, null, null, 25130180865, 15016964022, 17255588720, 39751154898, 4011295, "SRX13441048", "SRS11340477", "SRA1345465", "Bruce Draper Lab, Molecular and Cellular Biology, University of California, Davis", "Bruce Draper Lab, Molecular and Cellular Biology, University of California, Davis", 2, 0.00058, 0.83872, 0.00016, 0.05616, 0.99989, 0.77873, 0.4, 0.51521, 151, 151, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-17", "Juvenile", "Juvenile", "Gonad", "Reproductive System"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["67760"], "units": {}, "query_ms": 9.22628199623432}