metadata: run_metadata: 331 rows where experiment.library_strategy = "RNA-Seq", technology = "10x" and tissue

331 rows where experiment.library_strategy = "RNA-Seq", technology = "10x" and tissue_curation = "Eye"

descending

Link	rowid ▼	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	study.attributes	study.PMIDs	sample.title	sample.alias	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.extractprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.study_ref	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	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
25107	25107	SRR25605432	SRX21332628	SRS18578260	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep3	GSM7702835	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702835	GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq	GSM7702835 r1	GSM7702835	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_GFP_pos_S4_L001_R2_001.fastq.gz 2707_GFP_pos_S4_L001_R1_001.fastq.gz 2707_GFP_pos_S4_L001_I2_001.fastq.gz 2707_GFP_pos_S4_L001_I1_001.fastq.gz	fastq fastq fastq fastq	21603309774.0	156545723.0	GSM7702835 r1	0:10 1:10 2:28 3:90	A:3960451458;C:3105338977;G:3480072824;T:3541218595;N:2033216	10	10	28	90	3960451458	3105338977	3480072824	3541218595	2033216	SRX21332628	SRS18578260	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94286		0.14175		0.78395		0.51541		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25108	25108	SRR25605433	SRX21332628	SRS18578260	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep3	GSM7702835	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702835	GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq	GSM7702835 r1	GSM7702835	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_GFP_pos_S4_L002_I1_001.fastq.gz 2707_GFP_pos_S4_L002_I2_001.fastq.gz 2707_GFP_pos_S4_L002_R1_001.fastq.gz 2707_GFP_pos_S4_L002_R2_001.fastq.gz	fastq fastq fastq fastq	22413465270.0	162416415.0	GSM7702835 r2	0:10 1:10 2:28 3:90	A:4104405257;C:3220235633;G:3625448968;T:3667329471;N:58021	10	10	28	90	4104405257	3220235633	3625448968	3667329471	58021	SRX21332628	SRS18578260	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94239		0.14234		0.78338		0.50616		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25109	25109	SRR25822232	SRX21332628	SRS18578260	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep3	GSM7702835	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702835	GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq	GSM7702835 r1	GSM7702835	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_GFP_pos_S4_L001_R2_002.fastq.gz 2707_GFP_pos_S4_L001_R1_002.fastq.gz 2707_GFP_pos_S4_L001_I2_002.fastq.gz 2707_GFP_pos_S4_L001_I1_002.fastq.gz	fastq fastq fastq fastq	21424338264.0	155248828.0	GSM7702835 r3	0:10 1:10 2:28 3:90	A:3928077020;C:3077672525;G:3451667186;T:3513734857;N:1242932	10	10	28	90	3928077020	3077672525	3451667186	3513734857	1242932	SRX21332628	SRS18578260	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.9433		0.14242		0.78328		0.52384		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25110	25110	SRR25822233	SRX21332628	SRS18578260	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep3	GSM7702835	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702835	GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq	GSM7702835 r1	GSM7702835	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_GFP_pos_S4_L002_I1_002.fastq.gz 2707_GFP_pos_S4_L002_I2_002.fastq.gz 2707_GFP_pos_S4_L002_R1_002.fastq.gz 2707_GFP_pos_S4_L002_R2_002.fastq.gz	fastq fastq fastq fastq	22542479952.0	163351304.0	GSM7702835 r4	0:10 1:10 2:28 3:90	A:4130174287;C:3235362228;G:3643457599;T:3691295761;N:1327485	10	10	28	90	4130174287	3235362228	3643457599	3691295761	1327485	SRX21332628	SRS18578260	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94183		0.14124		0.78301		0.50517		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25111	25111	SRR25605434	SRX21332627	SRS18578259	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep3	GSM7702834	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702834	GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq	GSM7702834 r1	GSM7702834	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_pos_Mcherry_S3_L001_R2_001.fastq.gz 2707_pos_Mcherry_S3_L001_R1_001.fastq.gz 2707_pos_Mcherry_S3_L001_I2_001.fastq.gz 2707_pos_Mcherry_S3_L001_I1_001.fastq.gz	fastq fastq fastq fastq	20161522758.0	146097991.0	GSM7702834 r1	0:10 1:10 2:28 3:90	A:3649819934;C:2958286158;G:3267504300;T:3271384686;N:1824112	10	10	28	90	3649819934	2958286158	3267504300	3271384686	1824112	SRX21332627	SRS18578259	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94851		0.1483		0.80012		0.52549		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25112	25112	SRR25605435	SRX21332627	SRS18578259	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep3	GSM7702834	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702834	GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq	GSM7702834 r1	GSM7702834	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_pos_Mcherry_S3_L002_I1_001.fastq.gz 2707_pos_Mcherry_S3_L002_I2_001.fastq.gz 2707_pos_Mcherry_S3_L002_R1_001.fastq.gz 2707_pos_Mcherry_S3_L002_R2_001.fastq.gz	fastq fastq fastq fastq	20708273928.0	150059956.0	GSM7702834 r2	0:10 1:10 2:28 3:90	A:3746088654;C:3037032642;G:3368938151;T:3353289242;N:47351	10	10	28	90	3746088654	3037032642	3368938151	3353289242	47351	SRX21332627	SRS18578259	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94778		0.14581		0.80028		0.52885		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25113	25113	SRR25822230	SRX21332627	SRS18578259	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep3	GSM7702834	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702834	GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq	GSM7702834 r1	GSM7702834	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_pos_Mcherry_S3_L001_I1_002.fastq.gz 2707_pos_Mcherry_S3_L001_I2_002.fastq.gz 2707_pos_Mcherry_S3_L001_R1_002.fastq.gz 2707_pos_Mcherry_S3_L001_R2_002.fastq.gz	fastq fastq fastq fastq	20078121078.0	145493631.0	GSM7702834 r3	0:10 1:10 2:28 3:90	A:3634984131;C:2943891505;G:3255149272;T:3259243920;N:1157962	10	10	28	90	3634984131	2943891505	3255149272	3259243920	1157962	SRX21332627	SRS18578259	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94884		0.14622		0.80099		0.52572		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25114	25114	SRR25822231	SRX21332627	SRS18578259	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep3	GSM7702834	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702834	GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq	GSM7702834 r1	GSM7702834	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	2707_pos_Mcherry_S3_L002_I1_002.fastq.gz 2707_pos_Mcherry_S3_L002_I2_002.fastq.gz 2707_pos_Mcherry_S3_L002_R1_002.fastq.gz 2707_pos_Mcherry_S3_L002_R2_002.fastq.gz	fastq fastq fastq fastq	20884392978.0	151336181.0	GSM7702834 r4	0:10 1:10 2:28 3:90	A:3779370721;C:3059502508;G:3396295214;T:3383865877;N:1221970	10	10	28	90	3779370721	3059502508	3396295214	3383865877	1221970	SRX21332627	SRS18578259	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.94896		0.14559		0.8002		0.51418		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25115	25115	SRR25605436	SRX21332626	SRS18578258	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep2	GSM7702833	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702833	GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq	GSM7702833 r1	GSM7702833	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py\|options: allowEarlyFileEnd	1907_GFP_pos_S2_L001_I1_001.fastq.gz 1907_GFP_pos_S2_L001_I2_001.fastq.gz 1907_GFP_pos_S2_L001_R1_001.fastq.gz 1907_GFP_pos_S2_L001_R2_001.fastq.gz	fastq fastq fastq fastq	22174775364.0	160686778.0	GSM7702833 r1	0:10 1:10 2:28 3:90	A:4207510387;C:3050740898;G:3362227609;T:3839333241;N:1997885	10	10	28	90	4207510387	3050740898	3362227609	3839333241	1997885	SRX21332626	SRS18578258	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93287		0.11844		0.81087		0.55264		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25116	25116	SRR25605437	SRX21332626	SRS18578258	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep2	GSM7702833	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702833	GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq	GSM7702833 r1	GSM7702833	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py\|options: allowEarlyFileEnd	1907_GFP_pos_S2_L002_I1_001.fastq.gz 1907_GFP_pos_S2_L002_I2_001.fastq.gz 1907_GFP_pos_S2_L002_R1_001.fastq.gz 1907_GFP_pos_S2_L002_R2_001.fastq.gz	fastq fastq fastq fastq	22047847518.0	159767011.0	GSM7702833 r2	0:10 1:10 2:28 3:90	A:4174013532;C:3033959926;G:3357962867;T:3813042664;N:52001	10	10	28	90	4174013532	3033959926	3357962867	3813042664	52001	SRX21332626	SRS18578258	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93359		0.11585		0.81087		0.55705		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25117	25117	SRR25822228	SRX21332626	SRS18578258	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep2	GSM7702833	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702833	GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq	GSM7702833 r1	GSM7702833	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_GFP_pos_S2_L001_I1_002.fastq.gz 1907_GFP_pos_S2_L001_I2_002.fastq.gz 1907_GFP_pos_S2_L001_R1_002.fastq.gz 1907_GFP_pos_S2_L001_R2_002.fastq.gz	fastq fastq fastq fastq	22010894568.0	159499236.0	GSM7702833 r3	0:10 1:10 2:28 3:90	A:4176090595;C:3026940106;G:3336506731;T:3814128890;N:1264918	10	10	28	90	4176090595	3026940106	3336506731	3814128890	1264918	SRX21332626	SRS18578258	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93457		0.11733		0.81032		0.55203		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25118	25118	SRR25822229	SRX21332626	SRS18578258	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep2	GSM7702833	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702833	GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq	GSM7702833 r1	GSM7702833	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_GFP_pos_S2_L002_I1_002.fastq.gz 1907_GFP_pos_S2_L002_I2_002.fastq.gz 1907_GFP_pos_S2_L002_R1_002.fastq.gz 1907_GFP_pos_S2_L002_R2_002.fastq.gz	fastq fastq fastq fastq	22227220746.0	161066817.0	GSM7702833 r4	0:10 1:10 2:28 3:90	A:4209198596;C:3055507768;G:3382717459;T:3847294849;N:1294858	10	10	28	90	4209198596	3055507768	3382717459	3847294849	1294858	SRX21332626	SRS18578258	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93428		0.11629		0.81014		0.56014		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25119	25119	SRR25605438	SRX21332625	SRS18578257	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep2	GSM7702832	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702832	GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq	GSM7702832 r1	GSM7702832	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_pos_pos_S1_L001_I1_001.fastq.gz 1907_pos_pos_S1_L001_I2_001.fastq.gz 1907_pos_pos_S1_L001_R1_001.fastq.gz 1907_pos_pos_S1_L001_R2_001.fastq.gz	fastq fastq fastq fastq	25141955454.0	182188083.0	GSM7702832 r1	0:10 1:10 2:28 3:90	A:4942067104;C:3446689328;G:3778138998;T:4227670796;N:2361244	10	10	28	90	4942067104	3446689328	3778138998	4227670796	2361244	SRX21332625	SRS18578257	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.90803		0.11273		0.81872		0.54345		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25120	25120	SRR25605439	SRX21332625	SRS18578257	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep2	GSM7702832	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702832	GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq	GSM7702832 r1	GSM7702832	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_pos_pos_S1_L002_R2_001.fastq.gz 1907_pos_pos_S1_L002_R1_001.fastq.gz 1907_pos_pos_S1_L002_I2_001.fastq.gz 1907_pos_pos_S1_L002_I1_001.fastq.gz	fastq fastq fastq fastq	25433730786.0	184302397.0	GSM7702832 r2	0:10 1:10 2:28 3:90	A:4980326106;C:3488986366;G:3842594871;T:4275245265;N:63122	10	10	28	90	4980326106	3488986366	3842594871	4275245265	63122	SRX21332625	SRS18578257	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.91035		0.11175		0.82016		0.52814		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25121	25121	SRR25822226	SRX21332625	SRS18578257	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep2	GSM7702832	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702832	GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq	GSM7702832 r1	GSM7702832	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_pos_pos_S1_L001_R2_002.fastq.gz 1907_pos_pos_S1_L001_R1_002.fastq.gz 1907_pos_pos_S1_L001_I2_002.fastq.gz 1907_pos_pos_S1_L001_I1_002.fastq.gz	fastq fastq fastq fastq	24834656088.0	179961276.0	GSM7702832 r3	0:10 1:10 2:28 3:90	A:4878672093;C:3403443000;G:3732878174;T:4180051888;N:1469685	10	10	28	90	4878672093	3403443000	3732878174	4180051888	1469685	SRX21332625	SRS18578257	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.90776		0.11325		0.81852		0.54011		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25122	25122	SRR25822227	SRX21332625	SRS18578257	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep2	GSM7702832	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep2	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702832	GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq	GSM7702832 r1	GSM7702832	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	1907_pos_pos_S1_L002_I1_002.fastq.gz 1907_pos_pos_S1_L002_I2_002.fastq.gz 1907_pos_pos_S1_L002_R1_002.fastq.gz 1907_pos_pos_S1_L002_R2_002.fastq.gz	fastq fastq fastq fastq	25609437216.0	185575632.0	GSM7702832 r4	0:10 1:10 2:28 3:90	A:5015380345;C:3509305882;G:3867598814;T:4308011415;N:1510424	10	10	28	90	5015380345	3509305882	3867598814	4308011415	1510424	SRX21332625	SRS18578257	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.90907		0.11192		0.81994		0.50902		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25123	25123	SRR25605440	SRX21332624	SRS18578256	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep1	GSM7702831	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep1	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702831	GSM7702831: gfp enriched rep1; Danio rerio; RNA Seq	GSM7702831 r1	GSM7702831	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	gfp_nre_S1_L001_R2_001.fastq.gz gfp_nre_S1_L001_R1_001.fastq.gz gfp_nre_S1_L001_I2_001.fastq.gz gfp_nre_S1_L001_I1_001.fastq.gz	fastq fastq fastq fastq	47873499684.0	346909418.0	GSM7702831 r1	0:10 1:10 2:28 3:90	A:8912593789;C:6786299583;G:7771099171;T:7737013838;N:14841239	10	10	28	90	8912593789	6786299583	7771099171	7737013838	14841239	SRX21332624	SRS18578256	SRA1690580	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.90717		0.15854		0.80501		0.50927		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25124	25124	SRR25605441	SRX21332624	SRS18578256	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	gfp enriched rep1	GSM7702831	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep1	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702831	GSM7702831: gfp enriched rep1; Danio rerio; RNA Seq	GSM7702831 r1	GSM7702831	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	gfp_nre_S1_L002_I1_001.fastq.gz gfp_nre_S1_L002_I2_001.fastq.gz gfp_nre_S1_L002_R1_001.fastq.gz gfp_nre_S1_L002_R2_001.fastq.gz	fastq fastq fastq fastq	48659347860.0	352603970.0	GSM7702831 r2	0:10 1:10 2:28 3:90	A:9056524463;C:6888387179;G:7922807308;T:7854042793;N:12595557	10	10	28	90	9056524463	6888387179	7922807308	7854042793	12595557	SRX21332624	SRS18578256	SRA1690580	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.90541		0.15686		0.80543		0.51051		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25125	25125	SRR25605442	SRX21332623	SRS18578255	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep1	GSM7702830	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep1	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702830	GSM7702830: mcherry enriched rep1; Danio rerio; RNA Seq	GSM7702830 r1	GSM7702830	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	double_pos_S2_L001_R2_001.fastq.gz double_pos_S2_L001_R1_001.fastq.gz double_pos_S2_L001_I2_001.fastq.gz double_pos_S2_L001_I1_001.fastq.gz	fastq fastq fastq fastq	41371188084.0	299791218.0	GSM7702830 r1	0:10 1:10 2:28 3:90	A:7690531012;C:5787287634;G:6514950407;T:6975618202;N:12822365	10	10	28	90	7690531012	5787287634	6514950407	6975618202	12822365	SRX21332623	SRS18578255	SRA1690580	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93894		0.17677		0.79926		0.51122		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
25126	25126	SRR25605443	SRX21332623	SRS18578255	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867	mcherry enriched rep1	GSM7702830	source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	mcherry enriched rep1	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted…	Eye	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.	tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702830	GSM7702830: mcherry enriched rep1; Danio rerio; RNA Seq	GSM7702830 r1	GSM7702830	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	NextSeq 2000	SRP454539	loader:fastq load.py	double_pos_S2_L002_I1_001.fastq.gz double_pos_S2_L002_I2_001.fastq.gz double_pos_S2_L002_R1_001.fastq.gz double_pos_S2_L002_R2_001.fastq.gz	fastq fastq fastq fastq	42692812980.0	309368210.0	GSM7702830 r2	0:10 1:10 2:28 3:90	A:7933061913;C:5964617781;G:6744802923;T:7189579846;N:11076437	10	10	28	90	7933061913	5964617781	6744802923	7189579846	11076437	SRX21332623	SRS18578255	SRA1690580	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.93887		0.17578		0.79825		0.50169		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System
29174	29174	SRR27237224	SRX22915658	SRS19883761	SRP478464	PRJNA1053781	Single cell gene expression profie of developing photoreceptor cells in larval zebrafish	GSE250379	Other	Molecular underpinnings of vertebrate retinal differentiation and maturation are poorly understood particularly for non mammalian species. We generated single cell transcriptome data from the larval zebrafish retina and characterized gene expression diversity among photoreceptor subtypes and their progenitors. Overall design: GFP positive differentiating photoreceptor cells and bipolar cells were collected from 4 dpf larval transgenic zebrafish Tgcrx:EGFPstl887 using fluorescence activated cell sorting.	parent bioproject:PRJNA1050288	pubmed:39531499	retina scRNA seq	GSM7978132	source name:retina\|tissue:retina\|genotype:Tgcrx:EGFPstl887\|developmental stage:4 dpf\|geo loc name:missing\|collection date:missing	retina scRNA seq	Read alignment and initial quality control were performed using Cell Ranger software version 7.0.0 10X Genomics. Assembly: GRCz11 Supplementary files format and content: Tab separated value file and matrix file	retina	Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection eyes were stored in ice cold Hanks’ Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA 25 mM HEPES 1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter side scatter and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+ supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min washed with D PBS CMF centrifuged and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer and 5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3 platform 10X Genomics Pleasanton CA according to the manufacturer protocol.	tissue:retina\|genotype:Tgcrx:EGFPstl887\|developmental stage:4 dpf	GSM7978132	GSM7978132: retina scRNA seq; Danio rerio; RNA Seq	GSM7978132 r1	GSM7978132	1	Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection eyes were stored in ice cold Hanks' Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA 25 mM HEPES 1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter side scatter and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+ supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min washed with D PBS CMF centrifuged and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer and 5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3 platform 10X Genomics Pleasanton CA according to the manufacturer protocol.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP478464	loader:fastq load.py	crx.crx_S1_L003_R1_001.fastq.gz crx.crx_S1_L003_R2_001.fastq.gz	fastq fastq	45170138494.0	253764823.0	GSM7978132 r1	0:28 1:150	A:13826023801;C:9023674621;G:9590649474;T:12728940802;N:849796	28	150			13826023801	9023674621	9590649474	12728940802	849796	SRX22915658	SRS19883761	SRA1770358	Pathology and Immunology, Washington University School of Medicine	Pathology and Immunology, Washington University School of Medicine	2	0.0046	0.87155	0.00206	0.2263	0.99168	0.77784	0.30223	0.50112	28	150	T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2023-12-17	Larval	Larval	Eye	Sensory System
32194	32194	SRR29141332	SRX24663085	SRS21398383	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr preablation wildtype control day 5 RNA	GSM8287442	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr preablation wildtype control day 5 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287442	GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq	GSM8287442 r1	GSM8287442	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH182R_S1_L001_R1_001.fastq.gz TH182R_S1_L001_R2_001.fastq.gz	fastq fastq	13807554173.0	116029867.0	GSM8287442 r1	0:28 1:91	A:4079582572;C:2862550576;G:2918085175;T:3947000701;N:335149	28	91			4079582572	2862550576	2918085175	3947000701	335149	SRX24663085	SRS21398383	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32195	32195	SRR29141333	SRX24663085	SRS21398383	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr preablation wildtype control day 5 RNA	GSM8287442	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr preablation wildtype control day 5 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287442	GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq	GSM8287442 r1	GSM8287442	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH182R_S1_L002_R1_001.fastq.gz TH182R_S1_L002_R2_001.fastq.gz	fastq fastq	13803126064.0	115992656.0	GSM8287442 r2	0:28 1:91	A:4078989982;C:2861718905;G:2916062814;T:3946005912;N:348451	28	91			4078989982	2861718905	2916062814	3946005912	348451	SRX24663085	SRS21398383	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32196	32196	SRR29141334	SRX24663085	SRS21398383	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr preablation wildtype control day 5 RNA	GSM8287442	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr preablation wildtype control day 5 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287442	GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq	GSM8287442 r1	GSM8287442	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH182R_S1_L003_R1_001.fastq.gz TH182R_S1_L003_R2_001.fastq.gz	fastq fastq	14039364150.0	117977850.0	GSM8287442 r3	0:28 1:91	A:4148421178;C:2910249087;G:2967774612;T:4012572976;N:346297	28	91			4148421178	2910249087	2967774612	4012572976	346297	SRX24663085	SRS21398383	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32197	32197	SRR29141335	SRX24663085	SRS21398383	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr preablation wildtype control day 5 RNA	GSM8287442	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr preablation wildtype control day 5 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287442	GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq	GSM8287442 r1	GSM8287442	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH182R_S1_L004_R1_001.fastq.gz TH182R_S1_L004_R2_001.fastq.gz	fastq fastq	14396508973.0	120979067.0	GSM8287442 r4	0:28 1:91	A:4252694309;C:2985039203;G:3045994377;T:4112440375;N:340709	28	91			4252694309	2985039203	3045994377	4112440375	340709	SRX24663085	SRS21398383	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32198	32198	SRR29141336	SRX24663084	SRS21398382	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 7 24h mtz	GSM8287437	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 7 24h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287437	GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq	GSM8287437 r1	GSM8287437	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH111_S65_R1_001.fastq.gz TH111_S65_R2_001.fastq.gz	fastq fastq	10009743191.0	84115489.0	GSM8287437 r1	0:28 1:91	A:2899067167;C:2137099335;G:2274433670;T:2671820531;N:27322488	28	91			2899067167	2137099335	2274433670	2671820531	27322488	SRX24663084	SRS21398382	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32199	32199	SRR29141337	SRX24663084	SRS21398382	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 7 24h mtz	GSM8287437	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 7 24h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287437	GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq	GSM8287437 r1	GSM8287437	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH111_S66_R1_001.fastq.gz TH111_S66_R2_001.fastq.gz	fastq fastq	10310944686.0	86646594.0	GSM8287437 r2	0:28 1:91	A:2991162064;C:2200127755;G:2335919653;T:2755298440;N:28436774	28	91			2991162064	2200127755	2335919653	2755298440	28436774	SRX24663084	SRS21398382	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32200	32200	SRR29141338	SRX24663084	SRS21398382	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 7 24h mtz	GSM8287437	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 7 24h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287437	GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq	GSM8287437 r1	GSM8287437	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH111_S68_R1_001.fastq.gz TH111_S68_R2_001.fastq.gz	fastq fastq	13987726480.0	117543920.0	GSM8287437 r3	0:28 1:91	A:4045259735;C:2989556292;G:3181238633;T:3733480210;N:38191610	28	91			4045259735	2989556292	3181238633	3733480210	38191610	SRX24663084	SRS21398382	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32201	32201	SRR29141339	SRX24663084	SRS21398382	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 7 24h mtz	GSM8287437	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 7 24h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287437	GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq	GSM8287437 r1	GSM8287437	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH111_S67_R1_001.fastq.gz TH111_S67_R2_001.fastq.gz	fastq fastq	11876682069.0	99804051.0	GSM8287437 r4	0:28 1:91	A:3432787756;C:2538691346;G:2701757248;T:3170892876;N:32552843	28	91			3432787756	2538691346	2701757248	3170892876	32552843	SRX24663084	SRS21398382	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32202	32202	SRR29141340	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S61_L001_R1_001.fastq.gz TH136_S61_L001_R2_001.fastq.gz	fastq fastq	4774895706.0	40125174.0	GSM8287441 r1	0:28 1:91	A:1351265716;C:1042777184;G:1100567875;T:1280161557;N:123374	28	91			1351265716	1042777184	1100567875	1280161557	123374	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32203	32203	SRR29141341	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S64_L003_R1_001.fastq.gz TH136_S64_L003_R2_001.fastq.gz	fastq fastq	5538657579.0	46543341.0	GSM8287441 r10	0:28 1:91	A:1567217187;C:1211616483;G:1275241958;T:1484449584;N:132367	28	91			1567217187	1211616483	1275241958	1484449584	132367	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32204	32204	SRR29141342	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S64_L004_R1_001.fastq.gz TH136_S64_L004_R2_001.fastq.gz	fastq fastq	5403469057.0	45407303.0	GSM8287441 r11	0:28 1:91	A:1530355512;C:1180686287;G:1242924943;T:1449389493;N:112822	28	91			1530355512	1180686287	1242924943	1449389493	112822	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32205	32205	SRR29141343	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S61_L002_R1_001.fastq.gz TH136_S61_L002_R2_001.fastq.gz	fastq fastq	4786338627.0	40221333.0	GSM8287441 r12	0:28 1:91	A:1355122219;C:1044516973;G:1101760300;T:1284824360;N:114775	28	91			1355122219	1044516973	1101760300	1284824360	114775	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32206	32206	SRR29141344	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S62_L001_R1_001.fastq.gz TH136_S62_L001_R2_001.fastq.gz	fastq fastq	4901760535.0	41191265.0	GSM8287441 r13	0:28 1:91	A:1391714409;C:1068852387;G:1124767182;T:1316299153;N:127404	28	91			1391714409	1068852387	1124767182	1316299153	127404	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32207	32207	SRR29141345	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S62_L004_R1_001.fastq.gz TH136_S62_L004_R2_001.fastq.gz	fastq fastq	4937865968.0	41494672.0	GSM8287441 r14	0:28 1:91	A:1399404530;C:1078622301;G:1134489765;T:1325245845;N:103527	28	91			1399404530	1078622301	1134489765	1325245845	103527	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32208	32208	SRR29141346	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S63_L003_R1_001.fastq.gz TH136_S63_L003_R2_001.fastq.gz	fastq fastq	4497873940.0	37797260.0	GSM8287441 r15	0:28 1:91	A:1271859460;C:985043807;G:1034974096;T:1205889617;N:106960	28	91			1271859460	985043807	1034974096	1205889617	106960	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32209	32209	SRR29141347	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S64_L002_R1_001.fastq.gz TH136_S64_L002_R2_001.fastq.gz	fastq fastq	5236234787.0	44001973.0	GSM8287441 r16	0:28 1:91	A:1486231119;C:1141729846;G:1201608482;T:1406539690;N:125650	28	91			1486231119	1141729846	1201608482	1406539690	125650	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32210	32210	SRR29141348	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S61_L003_R1_001.fastq.gz TH136_S61_L003_R2_001.fastq.gz	fastq fastq	5047989877.0	42420083.0	GSM8287441 r2	0:28 1:91	A:1424658452;C:1105197562;G:1165585488;T:1352428003;N:120372	28	91			1424658452	1105197562	1165585488	1352428003	120372	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32211	32211	SRR29141349	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S61_L004_R1_001.fastq.gz TH136_S61_L004_R2_001.fastq.gz	fastq fastq	4922634682.0	41366678.0	GSM8287441 r3	0:28 1:91	A:1390880945;C:1076385249;G:1135254796;T:1320011695;N:101997	28	91			1390880945	1076385249	1135254796	1320011695	101997	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32212	32212	SRR29141350	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S62_L002_R1_001.fastq.gz TH136_S62_L002_R2_001.fastq.gz	fastq fastq	4903434746.0	41205334.0	GSM8287441 r4	0:28 1:91	A:1392700449;C:1068492247;G:1123677701;T:1318447188;N:117161	28	91			1392700449	1068492247	1123677701	1318447188	117161	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32213	32213	SRR29141351	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S62_L003_R1_001.fastq.gz TH136_S62_L003_R2_001.fastq.gz	fastq fastq	5079175136.0	42682144.0	GSM8287441 r5	0:28 1:91	A:1438032514;C:1110731492;G:1168141629;T:1362147196;N:122305	28	91			1438032514	1110731492	1168141629	1362147196	122305	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32214	32214	SRR29141352	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S63_L001_R1_001.fastq.gz TH136_S63_L001_R2_001.fastq.gz	fastq fastq	4248222293.0	35699347.0	GSM8287441 r6	0:28 1:91	A:1204282194;C:928219300;G:975617558;T:1139994829;N:108412	28	91			1204282194	928219300	975617558	1139994829	108412	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32215	32215	SRR29141353	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S63_L002_R1_001.fastq.gz TH136_S63_L002_R2_001.fastq.gz	fastq fastq	4256614411.0	35769869.0	GSM8287441 r7	0:28 1:91	A:1207165748;C:929375627;G:976155057;T:1143817351;N:100628	28	91			1207165748	929375627	976155057	1143817351	100628	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32216	32216	SRR29141354	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S63_L004_R1_001.fastq.gz TH136_S63_L004_R2_001.fastq.gz	fastq fastq	4376437534.0	36776786.0	GSM8287441 r8	0:28 1:91	A:1238798346;C:957263182;G:1005958070;T:1174327377;N:90559	28	91			1238798346	957263182	1005958070	1174327377	90559	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32217	32217	SRR29141355	SRX24663083	SRS21398381	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 9 72h mtz	GSM8287441	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 9 72h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287441	GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq	GSM8287441 r1	GSM8287441	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH136_S64_L001_R1_001.fastq.gz TH136_S64_L001_R2_001.fastq.gz	fastq fastq	5222497546.0	43886534.0	GSM8287441 r9	0:28 1:91	A:1481856778;C:1139695420;G:1200133908;T:1400676121;N:135319	28	91			1481856778	1139695420	1200133908	1400676121	135319	SRX24663083	SRS21398381	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32218	32218	SRR29141356	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S57_L001_R1_001.fastq.gz TH135_S57_L001_R2_001.fastq.gz	fastq fastq	4775761788.0	40132452.0	GSM8287440 r1	0:28 1:91	A:1364997508;C:1030536901;G:1087120578;T:1292982564;N:124237	28	91			1364997508	1030536901	1087120578	1292982564	124237	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32219	32219	SRR29141357	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S60_L002_R1_001.fastq.gz TH135_S60_L002_R2_001.fastq.gz	fastq fastq	5081243356.0	42699524.0	GSM8287440 r10	0:28 1:91	A:1453763884;C:1095418883;G:1154387721;T:1377550690;N:122178	28	91			1453763884	1095418883	1154387721	1377550690	122178	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32220	32220	SRR29141358	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S60_L004_R1_001.fastq.gz TH135_S60_L004_R2_001.fastq.gz	fastq fastq	5258119482.0	44185878.0	GSM8287440 r11	0:28 1:91	A:1501504527;C:1135881534;G:1197368112;T:1423254847;N:110462	28	91			1501504527	1135881534	1197368112	1423254847	110462	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32221	32221	SRR29141359	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S57_L003_R1_001.fastq.gz TH135_S57_L003_R2_001.fastq.gz	fastq fastq	5008964184.0	42092136.0	GSM8287440 r12	0:28 1:91	A:1428212274;C:1083563490;G:1142704874;T:1354362571;N:120975	28	91			1428212274	1083563490	1142704874	1354362571	120975	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32222	32222	SRR29141360	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S58_L002_R1_001.fastq.gz TH135_S58_L002_R2_001.fastq.gz	fastq fastq	5035503207.0	42315153.0	GSM8287440 r13	0:28 1:91	A:1440077950;C:1086334843;G:1144377517;T:1364592460;N:120437	28	91			1440077950	1086334843	1144377517	1364592460	120437	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32223	32223	SRR29141361	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S59_L001_R1_001.fastq.gz TH135_S59_L001_R2_001.fastq.gz	fastq fastq	5387099536.0	45269744.0	GSM8287440 r14	0:28 1:91	A:1537849058;C:1163912984;G:1227124886;T:1458073621;N:138987	28	91			1537849058	1163912984	1227124886	1458073621	138987	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32224	32224	SRR29141362	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S59_L004_R1_001.fastq.gz TH135_S59_L004_R2_001.fastq.gz	fastq fastq	5512521490.0	46323710.0	GSM8287440 r15	0:28 1:91	A:1571984524;C:1192238932;G:1256792838;T:1491389888;N:115308	28	91			1571984524	1192238932	1256792838	1491389888	115308	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32225	32225	SRR29141363	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S60_L003_R1_001.fastq.gz TH135_S60_L003_R2_001.fastq.gz	fastq fastq	5376169386.0	45177894.0	GSM8287440 r16	0:28 1:91	A:1533658425;C:1162739111;G:1225658907;T:1453983443;N:129500	28	91			1533658425	1162739111	1225658907	1453983443	129500	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32226	32226	SRR29141364	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S57_L002_R1_001.fastq.gz TH135_S57_L002_R2_001.fastq.gz	fastq fastq	4787380115.0	40230085.0	GSM8287440 r2	0:28 1:91	A:1368967190;C:1032261634;G:1088378612;T:1297658513;N:114166	28	91			1368967190	1032261634	1088378612	1297658513	114166	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32227	32227	SRR29141365	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S57_L004_R1_001.fastq.gz TH135_S57_L004_R2_001.fastq.gz	fastq fastq	4885123145.0	41051455.0	GSM8287440 r3	0:28 1:91	A:1394450278;C:1055564129;G:1113114189;T:1321891090;N:103459	28	91			1394450278	1055564129	1113114189	1321891090	103459	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32228	32228	SRR29141366	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S58_L001_R1_001.fastq.gz TH135_S58_L001_R2_001.fastq.gz	fastq fastq	5060757982.0	42527378.0	GSM8287440 r4	0:28 1:91	A:1446313544;C:1092878434;G:1151738503;T:1369695242;N:132259	28	91			1446313544	1092878434	1151738503	1369695242	132259	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32229	32229	SRR29141367	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S58_L003_R1_001.fastq.gz TH135_S58_L003_R2_001.fastq.gz	fastq fastq	5322898560.0	44730240.0	GSM8287440 r5	0:28 1:91	A:1517934750;C:1151967941;G:1213786668;T:1439081677;N:127524	28	91			1517934750	1151967941	1213786668	1439081677	127524	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32230	32230	SRR29141368	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S58_L004_R1_001.fastq.gz TH135_S58_L004_R2_001.fastq.gz	fastq fastq	5160850786.0	43368494.0	GSM8287440 r6	0:28 1:91	A:1473328102;C:1115516878;G:1175502159;T:1396395641;N:108006	28	91			1473328102	1115516878	1175502159	1396395641	108006	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32231	32231	SRR29141369	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S59_L002_R1_001.fastq.gz TH135_S59_L002_R2_001.fastq.gz	fastq fastq	5381504513.0	45222727.0	GSM8287440 r7	0:28 1:91	A:1537238062;C:1161721381;G:1224135102;T:1458280892;N:129076	28	91			1537238062	1161721381	1224135102	1458280892	129076	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32232	32232	SRR29141370	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S59_L003_R1_001.fastq.gz TH135_S59_L003_R2_001.fastq.gz	fastq fastq	5665367827.0	47608133.0	GSM8287440 r8	0:28 1:91	A:1613897652;C:1226812698;G:1293000098;T:1531521187;N:136192	28	91			1613897652	1226812698	1293000098	1531521187	136192	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32233	32233	SRR29141371	SRX24663082	SRS21398380	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Ablated rgc:ntr day 6 12h mtz	GSM8287440	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz\|geo loc name:missing\|collection date:missing	Ablated rgc:ntr day 6 12h mtz	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz	GSM8287440	GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq	GSM8287440 r1	GSM8287440	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH135_S60_L001_R1_001.fastq.gz TH135_S60_L001_R2_001.fastq.gz	fastq fastq	5045550377.0	42399583.0	GSM8287440 r9	0:28 1:91	A:1442885146;C:1088672739;G:1147630180;T:1366230521;N:131791	28	91			1442885146	1088672739	1147630180	1366230521	131791	SRX24663082	SRS21398380	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32234	32234	SRR29141372	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S53_L001_R1_001.fastq.gz TH134_S53_L001_R2_001.fastq.gz	fastq fastq	4566870378.0	38377062.0	GSM8287439 r1	0:28 1:91	A:1300193484;C:990777876;G:1047244494;T:1228536400;N:118124	28	91			1300193484	990777876	1047244494	1228536400	118124	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32235	32235	SRR29141373	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S55_L002_R1_001.fastq.gz TH134_S55_L002_R2_001.fastq.gz	fastq fastq	4872575309.0	40946011.0	GSM8287439 r10	0:28 1:91	A:1388373514;C:1056698672;G:1113330484;T:1314056163;N:116476	28	91			1388373514	1056698672	1113330484	1314056163	116476	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32236	32236	SRR29141374	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S55_L003_R1_001.fastq.gz TH134_S55_L003_R2_001.fastq.gz	fastq fastq	5138052528.0	43176912.0	GSM8287439 r11	0:28 1:91	A:1459926271;C:1117597394;G:1177808023;T:1382597761;N:123079	28	91			1459926271	1117597394	1177808023	1382597761	123079	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32237	32237	SRR29141375	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S55_L004_R1_001.fastq.gz TH134_S55_L004_R2_001.fastq.gz	fastq fastq	5022868263.0	42208977.0	GSM8287439 r12	0:28 1:91	A:1428914798;C:1091192534;G:1150147876;T:1352508063;N:104992	28	91			1428914798	1091192534	1150147876	1352508063	104992	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32238	32238	SRR29141376	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S56_L001_R1_001.fastq.gz TH134_S56_L001_R2_001.fastq.gz	fastq fastq	4569776953.0	38401487.0	GSM8287439 r13	0:28 1:91	A:1299842950;C:992232187;G:1048131188;T:1229452715;N:117913	28	91			1299842950	992232187	1048131188	1229452715	117913	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32239	32239	SRR29141377	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S56_L002_R1_001.fastq.gz TH134_S56_L002_R2_001.fastq.gz	fastq fastq	4604427135.0	38692665.0	GSM8287439 r14	0:28 1:91	A:1310481305;C:998823194;G:1054488359;T:1240523819;N:110458	28	91			1310481305	998823194	1054488359	1240523819	110458	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32240	32240	SRR29141378	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S56_L003_R1_001.fastq.gz TH134_S56_L003_R2_001.fastq.gz	fastq fastq	4894210580.0	41127820.0	GSM8287439 r15	0:28 1:91	A:1389263901;C:1064650503;G:1124176385;T:1316003245;N:116546	28	91			1389263901	1064650503	1124176385	1316003245	116546	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32241	32241	SRR29141379	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S56_L004_R1_001.fastq.gz TH134_S56_L004_R2_001.fastq.gz	fastq fastq	4782327494.0	40187626.0	GSM8287439 r16	0:28 1:91	A:1359054303;C:1039048484;G:1097178265;T:1286945985;N:100457	28	91			1359054303	1039048484	1097178265	1286945985	100457	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32242	32242	SRR29141380	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S53_L002_R1_001.fastq.gz TH134_S53_L002_R2_001.fastq.gz	fastq fastq	4587896012.0	38553748.0	GSM8287439 r2	0:28 1:91	A:1307093057;C:994483546;G:1050434019;T:1235776211;N:109179	28	91			1307093057	994483546	1050434019	1235776211	109179	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32243	32243	SRR29141381	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S53_L003_R1_001.fastq.gz TH134_S53_L003_R2_001.fastq.gz	fastq fastq	4853618728.0	40786712.0	GSM8287439 r3	0:28 1:91	A:1378590543;C:1055361040;G:1114790969;T:1304760000;N:116176	28	91			1378590543	1055361040	1114790969	1304760000	116176	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32244	32244	SRR29141382	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S53_L004_R1_001.fastq.gz TH134_S53_L004_R2_001.fastq.gz	fastq fastq	4736870803.0	39805637.0	GSM8287439 r4	0:28 1:91	A:1346988869;C:1028744736;G:1086568704;T:1274469646;N:98848	28	91			1346988869	1028744736	1086568704	1274469646	98848	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32245	32245	SRR29141383	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S54_L001_R1_001.fastq.gz TH134_S54_L001_R2_001.fastq.gz	fastq fastq	5319795040.0	44704160.0	GSM8287439 r5	0:28 1:91	A:1516665110;C:1153199308;G:1216078023;T:1433713315;N:139284	28	91			1516665110	1153199308	1216078023	1433713315	139284	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32246	32246	SRR29141384	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S54_L002_R1_001.fastq.gz TH134_S54_L002_R2_001.fastq.gz	fastq fastq	5330566682.0	44794678.0	GSM8287439 r6	0:28 1:91	A:1520480633;C:1154635844;G:1217037822;T:1438283496;N:128887	28	91			1520480633	1154635844	1217037822	1438283496	128887	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32247	32247	SRR29141385	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S54_L003_R1_001.fastq.gz TH134_S54_L003_R2_001.fastq.gz	fastq fastq	5561785705.0	46737695.0	GSM8287439 r7	0:28 1:91	A:1581477655;C:1208929874;G:1274395111;T:1496847765;N:135300	28	91			1581477655	1208929874	1274395111	1496847765	135300	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32248	32248	SRR29141386	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S54_L004_R1_001.fastq.gz TH134_S54_L004_R2_001.fastq.gz	fastq fastq	5423072403.0	45572037.0	GSM8287439 r8	0:28 1:91	A:1543580201;C:1177607853;G:1241277315;T:1460492382;N:114652	28	91			1543580201	1177607853	1241277315	1460492382	114652	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32249	32249	SRR29141387	SRX24663081	SRS21398379	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 9	GSM8287439	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 9	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287439	GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq	GSM8287439 r1	GSM8287439	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH134_S55_L001_R1_001.fastq.gz TH134_S55_L001_R2_001.fastq.gz	fastq fastq	4848214105.0	40741295.0	GSM8287439 r9	0:28 1:91	A:1380197121;C:1052472596;G:1109522284;T:1305896718;N:125386	28	91			1380197121	1052472596	1109522284	1305896718	125386	SRX24663081	SRS21398379	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32250	32250	SRR29141388	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S49_L001_R1_001.fastq.gz TH133_S49_L001_R2_001.fastq.gz	fastq fastq	4633962102.0	38940858.0	GSM8287438 r1	0:28 1:91	A:1297725412;C:1024809339;G:1076904995;T:1234401645;N:120711	28	91			1297725412	1024809339	1076904995	1234401645	120711	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32251	32251	SRR29141389	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S52_L003_R1_001.fastq.gz TH133_S52_L003_R2_001.fastq.gz	fastq fastq	4989425217.0	41927943.0	GSM8287438 r10	0:28 1:91	A:1391245093;C:1106917719;G:1165216341;T:1325928222;N:117842	28	91			1391245093	1106917719	1165216341	1325928222	117842	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32252	32252	SRR29141390	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S52_L004_R1_001.fastq.gz TH133_S52_L004_R2_001.fastq.gz	fastq fastq	4854872155.0	40797245.0	GSM8287438 r11	0:28 1:91	A:1355524409;C:1075673845;G:1132295861;T:1291276543;N:101497	28	91			1355524409	1075673845	1132295861	1291276543	101497	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32253	32253	SRR29141391	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S49_L002_R1_001.fastq.gz TH133_S49_L002_R2_001.fastq.gz	fastq fastq	4657232314.0	39136406.0	GSM8287438 r12	0:28 1:91	A:1305512663;C:1028718382;G:1080733287;T:1242157091;N:110891	28	91			1305512663	1028718382	1080733287	1242157091	110891	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32254	32254	SRR29141392	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S50_L001_R1_001.fastq.gz TH133_S50_L001_R2_001.fastq.gz	fastq fastq	4932588794.0	41450326.0	GSM8287438 r13	0:28 1:91	A:1379015030;C:1091338503;G:1149356127;T:1312752653;N:126481	28	91			1379015030	1091338503	1149356127	1312752653	126481	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32255	32255	SRR29141393	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S50_L004_R1_001.fastq.gz TH133_S50_L004_R2_001.fastq.gz	fastq fastq	5077097277.0	42664683.0	GSM8287438 r14	0:28 1:91	A:1418123656;C:1124378298;G:1183528228;T:1350962964;N:104131	28	91			1418123656	1124378298	1183528228	1350962964	104131	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32256	32256	SRR29141394	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S51_L003_R1_001.fastq.gz TH133_S51_L003_R2_001.fastq.gz	fastq fastq	4923689498.0	41375542.0	GSM8287438 r15	0:28 1:91	A:1375204406;C:1090796811;G:1149080188;T:1308491701;N:116392	28	91			1375204406	1090796811	1149080188	1308491701	116392	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32257	32257	SRR29141395	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S52_L002_R1_001.fastq.gz TH133_S52_L002_R2_001.fastq.gz	fastq fastq	4696383909.0	39465411.0	GSM8287438 r16	0:28 1:91	A:1313674940;C:1038514326;G:1093116030;T:1250967700;N:110913	28	91			1313674940	1038514326	1093116030	1250967700	110913	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32258	32258	SRR29141396	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S49_L003_R1_001.fastq.gz TH133_S49_L003_R2_001.fastq.gz	fastq fastq	4935093863.0	41471377.0	GSM8287438 r2	0:28 1:91	A:1378860058;C:1094061046;G:1149630737;T:1312423104;N:118918	28	91			1378860058	1094061046	1149630737	1312423104	118918	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32259	32259	SRR29141397	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S49_L004_R1_001.fastq.gz TH133_S49_L004_R2_001.fastq.gz	fastq fastq	4820025147.0	40504413.0	GSM8287438 r3	0:28 1:91	A:1348394661;C:1067282812;G:1121376379;T:1282869478;N:101817	28	91			1348394661	1067282812	1121376379	1282869478	101817	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32260	32260	SRR29141398	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S50_L002_R1_001.fastq.gz TH133_S50_L002_R2_001.fastq.gz	fastq fastq	4931460079.0	41440841.0	GSM8287438 r4	0:28 1:91	A:1379937889;C:1089994166;G:1147241148;T:1314171218;N:115658	28	91			1379937889	1089994166	1147241148	1314171218	115658	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32261	32261	SRR29141399	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S50_L003_R1_001.fastq.gz TH133_S50_L003_R2_001.fastq.gz	fastq fastq	5229270193.0	43943447.0	GSM8287438 r5	0:28 1:91	A:1458651163;C:1159571989;G:1220750194;T:1390173862;N:122985	28	91			1458651163	1159571989	1220750194	1390173862	122985	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32262	32262	SRR29141400	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S51_L001_R1_001.fastq.gz TH133_S51_L001_R2_001.fastq.gz	fastq fastq	4636122190.0	38959010.0	GSM8287438 r6	0:28 1:91	A:1298955001;C:1023834288;G:1079293988;T:1233918757;N:120156	28	91			1298955001	1023834288	1079293988	1233918757	120156	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32263	32263	SRR29141401	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S51_L002_R1_001.fastq.gz TH133_S51_L002_R2_001.fastq.gz	fastq fastq	4651974894.0	39092226.0	GSM8287438 r7	0:28 1:91	A:1304274716;C:1026606682;G:1081394365;T:1239588507;N:110624	28	91			1304274716	1026606682	1081394365	1239588507	110624	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32264	32264	SRR29141402	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S51_L004_R1_001.fastq.gz TH133_S51_L004_R2_001.fastq.gz	fastq fastq	4797101463.0	40311777.0	GSM8287438 r8	0:28 1:91	A:1341600660;C:1061361282;G:1117958236;T:1276080791;N:100494	28	91			1341600660	1061361282	1117958236	1276080791	100494	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32265	32265	SRR29141403	SRX24663080	SRS21398378	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 6	GSM8287438	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 6	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287438	GSM8287438: Unablated rgc:ntr day 6; Danio rerio; RNA Seq	GSM8287438 r1	GSM8287438	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH133_S52_L001_R1_001.fastq.gz TH133_S52_L001_R2_001.fastq.gz	fastq fastq	4697283787.0	39472973.0	GSM8287438 r9	0:28 1:91	A:1312647560;C:1039809509;G:1095056786;T:1249648802;N:121130	28	91			1312647560	1039809509	1095056786	1249648802	121130	SRX24663080	SRS21398378	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32266	32266	SRR29141404	SRX24663079	SRS21398377	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 7	GSM8287436	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 7	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287436	GSM8287436: Unablated rgc:ntr day 7; Danio rerio; RNA Seq	GSM8287436 r1	GSM8287436	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH110_S62_R1_001.fastq.gz TH110_S62_R2_001.fastq.gz	fastq fastq	7832538588.0	65819652.0	GSM8287436 r1	0:28 1:91	A:2264636103;C:1687265841;G:1826367106;T:2032749605;N:21519933	28	91			2264636103	1687265841	1826367106	2032749605	21519933	SRX24663079	SRS21398377	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32267	32267	SRR29141405	SRX24663079	SRS21398377	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 7	GSM8287436	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 7	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287436	GSM8287436: Unablated rgc:ntr day 7; Danio rerio; RNA Seq	GSM8287436 r1	GSM8287436	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH110_S63_R1_001.fastq.gz TH110_S63_R2_001.fastq.gz	fastq fastq	166170767.0	1396393.0	GSM8287436 r2	0:28 1:91	A:48248797;C:35717941;G:38752344;T:42997083;N:454602	28	91			48248797	35717941	38752344	42997083	454602	SRX24663079	SRS21398377	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32268	32268	SRR29141406	SRX24663079	SRS21398377	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 7	GSM8287436	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 7	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287436	GSM8287436: Unablated rgc:ntr day 7; Danio rerio; RNA Seq	GSM8287436 r1	GSM8287436	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH110_S61_R1_001.fastq.gz TH110_S61_R2_001.fastq.gz	fastq fastq	8485930460.0	71310340.0	GSM8287436 r3	0:28 1:91	A:2462428503;C:1828880092;G:1970877358;T:2200821838;N:22922669	28	91			2462428503	1828880092	1970877358	2200821838	22922669	SRX24663079	SRS21398377	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32269	32269	SRR29141407	SRX24663079	SRS21398377	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Unablated rgc:ntr day 7	GSM8287436	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz\|geo loc name:missing\|collection date:missing	Unablated rgc:ntr day 7	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:no mtz	GSM8287436	GSM8287436: Unablated rgc:ntr day 7; Danio rerio; RNA Seq	GSM8287436 r1	GSM8287436	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	TH110_S64_R1_001.fastq.gz TH110_S64_R2_001.fastq.gz	fastq fastq	16842320614.0	141532106.0	GSM8287436 r4	0:28 1:91	A:5877167251;C:2818424553;G:4717101041;T:3385003561;N:44624208	28	91			5877167251	2818424553	4717101041	3385003561	44624208	SRX24663079	SRS21398377	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32270	32270	SRR29141408	SRX24663078	SRS21398376	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	GSM8287448	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs	GSM8287448	GSM8287448: Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA; Danio rerio; RNA Seq	GSM8287448 r1	GSM8287448	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	JM-M-R_S5_L001_R1_001.fastq.gz JM-M-R_S5_L001_R2_001.fastq.gz	fastq fastq	16128997724.0	135537796.0	GSM8287448 r1	0:28 1:91	A:4730995900;C:3379548236;G:3457738129;T:4560498122;N:217337	28	91			4730995900	3379548236	3457738129	4560498122	217337	SRX24663078	SRS21398376	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32271	32271	SRR29141409	SRX24663078	SRS21398376	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	GSM8287448	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs	GSM8287448	GSM8287448: Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA; Danio rerio; RNA Seq	GSM8287448 r1	GSM8287448	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	JM-M-R_S5_L002_R1_001.fastq.gz JM-M-R_S5_L002_R2_001.fastq.gz	fastq fastq	16319960475.0	137142525.0	GSM8287448 r2	0:28 1:91	A:4785034443;C:3421478325;G:3500876324;T:4612346844;N:224539	28	91			4785034443	3421478325	3500876324	4612346844	224539	SRX24663078	SRS21398376	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System
32272	32272	SRR29141410	SRX24663078	SRS21398376	SRP509393	PRJNA1115053	Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific	GSE268179	Other	Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.		pubmed:39007397	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	GSM8287448	source name:Eye\|tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs\|geo loc name:missing\|collection date:missing	Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA	scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files	Eye	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	tissue:Eye\|transgenic line:rgc:ntr\|treatment:mtz ascl1a gRNAs	GSM8287448	GSM8287448: Multiome rgc:ntr 24h ablation ascl1a KO day 7 RNA; Danio rerio; RNA Seq	GSM8287448 r1	GSM8287448	1	scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.	RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000	SRP509393	loader:fastq load.py	JM-M-R_S5_L003_R1_001.fastq.gz JM-M-R_S5_L003_R2_001.fastq.gz	fastq fastq	16272530526.0	136743954.0	GSM8287448 r3	0:28 1:91	A:4772770822;C:3409933317;G:3489014394;T:4600600715;N:211278	28	91			4772770822	3409933317	3489014394	4600600715	211278	SRX24663078	SRS21398376	SRA1875751	Jeff Mumm, Ophthalmology, Johns Hopkins University	Jeff Mumm, Ophthalmology, Johns Hopkins University												T	B	sc-like readlen	illumina	novaseq_era	unknown	cdna_unspecified	unknown	sc	single_cell_droplet	10x	United States	2024-05-23	Undetermined	Larval	Eye	Sensory System

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;