run_metadata: 67759

This data as json

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
67759	SRR17262951	SRX13441049	SRS11340478	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		pubmed:35588359		40 dpf zebrafish ovaries cells zx2	GSM5739895		tissue:40 dpf zebrafish ovaries\|cell type:Ovary cells\|genotype:AB wildtype	40 dpf zebrafish ovaries cells zx2	A General Transfer Format GTF gene annotation filerelease 96 for the GRCz11 zebrafish genome was downloaded from Ensembl Genome Browser and filtered using the “mkgtf” 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’s “mkref” 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 “count” function in Cell Ranger v3.0.2; 10x Genomic. “expect cells” was setted to 10000 based on estimated cell recovery. 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		Somatic ovary cells were dissociated using a modification of Elkouby and Mullins 2016. 40 pairs of 40 dpf ovaries were dissected from Tgpiwil1: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. post 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.	Zebrafish husbandry was performed as previously described Westerfield 2000.	cell type:Ovary cells\|genotype:AB wildtype	GSM5739895	GSM5739895: 40 dpf zebrafish ovaries cells zx2; Danio rerio; ssRNA seq	GSM5739895 r1	GSM5739895	1	Somatic ovary cells were dissociated using a modification of Elkouby and Mullins 2016. 40 pairs of 40 dpf ovaries were dissected from Tgpiwil1: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. post 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 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.		RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina HiSeq 4000		SRP351377			40dpf_ova_1_S146_L004_R2_001.fastq.gz 40dpf_ova_1_S146_L004_R1_001.fastq.gz	fastq fastq	109753922368.0	363423584.0	GSM5739895 r1	0:151 1:151	A:26550231612;C:18985464266;G:18837331344;T:45367377509;N:13517637	151	151			26550231612	18985464266	18837331344	45367377509	13517637	SRX13441049	SRS11340478	SRA1345465	Bruce Draper Lab, Molecular and Cellular Biology, University of California, Davis	Bruce Draper Lab, Molecular and Cellular Biology, University of California, Davis	2	9e-05	0.84548	8e-05	0.09287	1.0	0.78589		0.54454	151	151	T	B	mate1 technical by mapping diff	illumina	hiseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x		United States	2021-12-17	Juvenile	Juvenile	Gonad	Reproductive System