run_metadata: 64414

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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
64414	SRR14702158	SRX11040181	SRS9109179	SRP322166	PRJNA734313	Single cell profiling of photoreceptor cells in adult zebrafish	GSE175929	Other	Vertebrate vision is mediated by two kinds of photoreceptors rods and cones responsible for dim and bright light vision respectively. Gene expression differences among cone subtypes remain poorly understood compared with rods. We generated single cell transcriptome data using a droplet based approach to reveal the extent of gene expression diversity among adult zebrafish photoreceptor subtypes. Populations of photoreceptor cells were enriched by using the transgenic zebrafish lines Tgrho:EGFPja2Tg and Tggnat2:EGFPja23Tg which express GFP in rods and all cone subtypes respectively. By analyzing the single cell transcriptomes we found that in addition to the four canonical zebrafish cone types ultraviolet blue green and red there exist subpopulations of green and red cones in the ventral retina that express red shifted opsin paralogs opn1mw4 and opn1lw1. This work lays a foundation for future studies aimed at understanding how molecular differences among cone subtypes affect photoreceptor function. Overall design: GFP positive cells collected from two transgenic zebrafish Tgrho:EGFPja2Tg and Tggnat2:EGFPja23Tg were combined into one sample. A small percentage of GFP negative cells was also included in the sample.		pubmed:34462505		retinal cells	GSM5351368		tissue:retinal cells\|age:adult\|transgene:Tgrho:egfpja2Tg\|transgene:Tggnat2:egfpja23Tg	retinal cells	FASTQ files were generated from the raw BCL files using Illumina’s bcl2fastq conversion program. Sample demultiplexing with the mkfastq function from Cell Ranger softwareversion 3.1.0 10X Genomics. Sample alignment with the count function from Cell Ranger software version 6.0.0 10X Genomics. Paired end reads were mapped to the zebrafish genome GRCz11 using Cell Ranger software version 6.0.0 10X Genomics. Mapped reads were quantified using the Cell Ranger software with a custom annotation v432 dr adult eye.gtf included as processed file. The GFP transcript sequence was added manually to the reference assembly as an extra chromosome. Data were analyzed using the Seurat R package v4.0.0. We retained all cells that expressed >500 genes and we considered all genes expressed in at least five cells. Cells with greater than 30% mitochondrial gene content or >40 000 unique molecular identifiers were removed from the analysis. For the remaining cells a gene expression matrix was normalized to total cellular read counts using the Seurat SCTransform function. The 3 000 most variable genes identified by the SCTransform function were used for Principal Component Analysis. The top 30 principal components were selected for subsequent analysis. Graph based clustering was performed to obtain a set of transcriptionally distinct clusters. At this point in the analysis we deliberately set parameters to "over cluster" the data to avoid combining distinct cell types and to identify sub populations of low quality cells for removal. To retain high quality photoreceptors and bipolar cells only we subjected our data to multiple rounds of clustering filtering and selection. In the first round we retained those clusters characterized either by the presence of one or more of the following opsin genes or phototransduction genes rho opn1sw1 opn1sw2 opn1mw1 opn1mw2 opn1mw3 opn1mw4 opn1lw1 opn1lw2 gnat1 or gnat2 or bipolar specific genes e.g. gnao1b vsx1 cabp2a cabp5a and cabp5b among the top 20 most differentially expressed genes as identified by the FindAllMarkers function in Seurat. We also removed clusters consisting of low quality cells with low total gene counts 500 1 000 genes/cell compared with high quality photoreceptor clusters 1 000 3 000 genes/cell for rods and 1 000 4 000 genes/cell for cones. In the second round of clustering and selection we removed clusters that showed co expression of photoreceptor genes and Müller glial genes icn fxyd6l mt2 rlbp1a and glula. We also removed one cluster showing co expression of photoreceptor and bipolar genes and with low total gene counts. In the final round of clustering and selection we removed a rod subpopulation with low total gene counts and retained 2 186 high quality cells. Genome build: GRCz11 Supplementary files format and content: The Gene transfer format GTF containing information about gene structure used for counting the transcripts in the data Supplementary files format and content: Seurat object for scRNA seq data	retinal cells		Retinas of five mpf adult zebrafish were were harvested at around zeitgeber time 3 ZT 3. The dissected retinas were dissociated with a papain dissociation solution followed by further incubation with 10% FBS in DMEM and DNaseI. post the trituration the samples were resuspended in sorting buffer 20 mM HEPES and 0.04% bovine serum albumin in Calcium and magnesium free HBSS pH 7.4. Four retinas from two individuals were combined for Tgrho:EGFP fish while six retinas from three individuals were combined for Tggnat2:EGFP fish. The filtered samples were incubated in a solution of propidium iodide and Hoechst 33342. GFP positive cells were isolated via fluorescence activated cell sorting FACS. Dead cells were then removed based on propidium iodide positivity. Intact rods and cones were then selected based on the presence of both blue Hoechst 33342 and green fluorescence GFP. About 35 000 viable intact GFP positive cells PI GFP+ Hoechst+ and 1 500 GFP negative cells PI GFP Hoechst+ were collected from Tggnat2:EGFP fish while 10 000 GFP viable intact GFP positive cells and 1 500 GFP negative cells were collected from Tgrho:EGFP fish. These isolated cells were collected into the sorting buffer in one tube. Approximately 6 000 single cells were loaded into a 10X Chromium Single Cell Chip. Single cell libraries were made with Chromium 3’ v3 platform following the manufacturer’s protocol. RNA libraries were prepared for sequencing using standard Illumina protocols.		age:adult\|transgene:Tgrho:egfpja2Tg\|transgene:Tggnat2:egfpja23Tg	GSM5351368	GSM5351368: retinal cells; Danio rerio; RNA Seq	GSM5351368		1	Retinas of five mpf adult zebrafish were were harvested at around zeitgeber time 3 ZT 3. The dissected retinas were dissociated with a papain dissociation solution followed by further incubation with 10% FBS in DMEM and DNaseI. post the trituration the samples were resuspended in sorting buffer 20 mM HEPES and 0.04% bovine serum albumin in Calcium and magnesium free HBSS pH 7.4. Four retinas from two individuals were combined for Tgrho:EGFP fish while six retinas from three individuals were combined for Tggnat2:EGFP fish. The filtered samples were incubated in a solution of propidium iodide and Hoechst 33342. GFP positive cells were isolated via fluorescence activated cell sorting FACS. Dead cells were then removed based on propidium iodide positivity. Intact rods and cones were then selected based on the presence of both blue Hoechst 33342 and green fluorescence GFP. About 35 000 viable intact GFP positive cells PI GFP+ Hoechst+ and 1 500 GFP negative cells PI GFP Hoechst+ were collected from Tggnat2:EGFP fish while 10 000 GFP viable intact GFP positive cells and 1 500 GFP negative cells were collected from Tgrho:EGFP fish. These isolated cells were collected into the sorting buffer in one tube. Approximately 6 000 single cells were loaded into a 10X Chromium Single Cell Chip. Single cell libraries were made with Chromium three prime v3 platform following the manufacturer's protocol. RNA libraries were prepared for sequencing using standard Illumina protocols.	GEO Accession:GSM5351368	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000		SRP322166			Cone_S5_L003_R1_001.fastq.gz Cone_S5_L003_R2_001.fastq.gz	fastq fastq	68659489710.0	544916585.0	GSM5351368 r1	0:28 1:98	A:19580055351;C:15466801940;G:15266233048;T:18341055919;N:5343452	28	98			19580055351	15466801940	15266233048	18341055919	5343452	SRX11040181	SRS9109179	SRA1239213	GEO	Pathology and Immunology, Washington University School of Medicine	2	0.0102	0.85996	0.00306	0.14003	0.98977	0.84595	0.44136	0.62184	28	98	T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x		United States	2021-06-01	Adult	Adult	Eye	Sensory System