{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_layout = \"SINGLE\", experiment.library_selection = \"cDNA\" and tissue_curation = \"Eye\"", "rows": [[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.", null, "pubmed:37643867", null, "gfp enriched rep3", "GSM7702835", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14175, null, 0.78395, null, 0.51541, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep3", "GSM7702835", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14234, null, 0.78338, null, 0.50616, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep3", "GSM7702835", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14242, null, 0.78328, null, 0.52384, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep3", "GSM7702835", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14124, null, 0.78301, null, 0.50517, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep3", "GSM7702834", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.1483, null, 0.80012, null, 0.52549, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep3", "GSM7702834", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14581, null, 0.80028, null, 0.52885, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep3", "GSM7702834", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14622, null, 0.80099, null, 0.52572, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep3", "GSM7702834", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.14559, null, 0.8002, null, 0.51418, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep2", "GSM7702833", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11844, null, 0.81087, null, 0.55264, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep2", "GSM7702833", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11585, null, 0.81087, null, 0.55705, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep2", "GSM7702833", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11733, null, 0.81032, null, 0.55203, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "gfp enriched rep2", "GSM7702833", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11629, null, 0.81014, null, 0.56014, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep2", "GSM7702832", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11273, null, 0.81872, null, 0.54345, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep2", "GSM7702832", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11175, null, 0.82016, null, 0.52814, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep2", "GSM7702832", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11325, null, 0.81852, null, 0.54011, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [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.", null, "pubmed:37643867", null, "mcherry enriched rep2", "GSM7702832", null, "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 \u2265 500  library size \u2265 800  and mitochondrial reads \u2264 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 \u2018vars.to.regress\u2019. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the \u2018s.features\u2019 and \u2018g2m.features\u2019 genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file", "Eye", null, "NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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\u2019 gene expression technology v3.1.", null, "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\u201330 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\u00b0C  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 \u00b5l FACSmax  the samples were passed through a 35 \u00b5m 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.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 2000", null, "SRP454539", null, "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, null, 0.11192, null, 0.81994, null, 0.50902, null, 90, null, "B", null, "usable mapping rate", "illumina", "nextseq_v2", "3prime", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-08-10", "Hatching", "Embryo", "Eye", "Sensory System"], [34807, "SRR32289929", "SRX27626974", "SRS24034243", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 6", "GSM8784941", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784941", "GSM8784941: FAC sorted RGCs at 14dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784941 r1", "GSM8784941", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122375.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 455140014.0, 8924314.0, "GSM8784941 r1", "0:51", "A:121395551;C:104372745;G:106322577;T:123006247;N:42894", 51, null, null, null, 121395551, 104372745, 106322577, 123006247, 42894, "SRX27626974", "SRS24034243", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34808, "SRR32289930", "SRX27626974", "SRS24034243", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 6", "GSM8784941", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784941", "GSM8784941: FAC sorted RGCs at 14dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784941 r1", "GSM8784941", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122375.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 466246080.0, 9142080.0, "GSM8784941 r2", "0:51", "A:124363664;C:106868642;G:108780226;T:126189949;N:43599", 51, null, null, null, 124363664, 106868642, 108780226, 126189949, 43599, "SRX27626974", "SRS24034243", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34809, "SRR32289931", "SRX27626973", "SRS24034241", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 5", "GSM8784940", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784940", "GSM8784940: FAC sorted RGCs at 14dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784940 r1", "GSM8784940", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121325.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 453422589.0, 8890639.0, "GSM8784940 r1", "0:51", "A:119318402;C:105882527;G:107813042;T:120365525;N:43093", 51, null, null, null, 119318402, 105882527, 107813042, 120365525, 43093, "SRX27626973", "SRS24034241", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34810, "SRR32289932", "SRX27626973", "SRS24034241", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 5", "GSM8784940", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784940", "GSM8784940: FAC sorted RGCs at 14dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784940 r1", "GSM8784940", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121325.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 464612040.0, 9110040.0, "GSM8784940 r2", "0:51", "A:122313464;C:108440796;G:110356081;T:123457974;N:43725", 51, null, null, null, 122313464, 108440796, 110356081, 123457974, 43725, "SRX27626973", "SRS24034241", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34811, "SRR32289933", "SRX27626972", "SRS24034242", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 4", "GSM8784939", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784939", "GSM8784939: FAC sorted RGCs at 14dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784939 r1", "GSM8784939", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121324.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 399194493.0, 7827343.0, "GSM8784939 r1", "0:51", "A:104203040;C:94204942;G:95935068;T:104813749;N:37694", 51, null, null, null, 104203040, 94204942, 95935068, 104813749, 37694, "SRX27626972", "SRS24034242", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34812, "SRR32289934", "SRX27626972", "SRS24034242", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 4", "GSM8784939", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784939", "GSM8784939: FAC sorted RGCs at 14dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784939 r1", "GSM8784939", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121324.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 407414775.0, 7988525.0, "GSM8784939 r2", "0:51", "A:106355289;C:96099106;G:97825197;T:107096823;N:38360", 51, null, null, null, 106355289, 96099106, 97825197, 107096823, 38360, "SRX27626972", "SRS24034242", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34813, "SRR32289935", "SRX27626971", "SRS24034240", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 3", "GSM8784938", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784938", "GSM8784938: FAC sorted RGCs at 14dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784938 r1", "GSM8784938", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121323.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 393819042.0, 7721942.0, "GSM8784938 r1", "0:51", "A:104716428;C:90712029;G:92875673;T:105476998;N:37914", 51, null, null, null, 104716428, 90712029, 92875673, 105476998, 37914, "SRX27626971", "SRS24034240", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34814, "SRR32289936", "SRX27626971", "SRS24034240", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 3", "GSM8784938", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784938", "GSM8784938: FAC sorted RGCs at 14dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784938 r1", "GSM8784938", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121323.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 404191065.0, 7925315.0, "GSM8784938 r2", "0:51", "A:107484459;C:93060010;G:95238340;T:108369497;N:38759", 51, null, null, null, 107484459, 93060010, 95238340, 108369497, 38759, "SRX27626971", "SRS24034240", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34815, "SRR32289937", "SRX27626970", "SRS24034239", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 2", "GSM8784937", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784937", "GSM8784937: FAC sorted RGCs at 14dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784937 r1", "GSM8784937", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121322.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 387042774.0, 7589074.0, "GSM8784937 r1", "0:51", "A:107517102;C:84333044;G:85962742;T:109191597;N:38289", 51, null, null, null, 107517102, 84333044, 85962742, 109191597, 38289, "SRX27626970", "SRS24034239", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34816, "SRR32289938", "SRX27626970", "SRS24034239", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 2", "GSM8784937", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784937", "GSM8784937: FAC sorted RGCs at 14dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784937 r1", "GSM8784937", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121322.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 399657012.0, 7836412.0, "GSM8784937 r2", "0:51", "A:111048661;C:86998697;G:88636677;T:112933522;N:39455", 51, null, null, null, 111048661, 86998697, 88636677, 112933522, 39455, "SRX27626970", "SRS24034239", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34817, "SRR32289939", "SRX27626969", "SRS24034236", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 1", "GSM8784936", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784936", "GSM8784936: FAC sorted RGCs at 14dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784936 r1", "GSM8784936", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121320.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 358289280.0, 7025280.0, "GSM8784936 r1", "0:51", "A:101071342;C:75109849;G:76553150;T:105519857;N:35082", 51, null, null, null, 101071342, 75109849, 76553150, 105519857, 35082, "SRX27626969", "SRS24034236", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34818, "SRR32289940", "SRX27626969", "SRS24034236", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 14dpi biological replicate 1", "GSM8784936", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 14dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784936", "GSM8784936: FAC sorted RGCs at 14dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784936 r1", "GSM8784936", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121320.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 370202625.0, 7258875.0, "GSM8784936 r2", "0:51", "A:104421674;C:77537410;G:79038293;T:109169570;N:35678", 51, null, null, null, 104421674, 77537410, 79038293, 109169570, 35678, "SRX27626969", "SRS24034236", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34819, "SRR32289941", "SRX27626968", "SRS24034237", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 5", "GSM8784935", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784935", "GSM8784935: FAC sorted RGCs at 10dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784935 r1", "GSM8784935", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122374.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 403359306.0, 7909006.0, "GSM8784935 r1", "0:51", "A:104505636;C:95925686;G:97605050;T:105285164;N:37770", 51, null, null, null, 104505636, 95925686, 97605050, 105285164, 37770, "SRX27626968", "SRS24034237", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34820, "SRR32289942", "SRX27626968", "SRS24034237", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 5", "GSM8784935", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784935", "GSM8784935: FAC sorted RGCs at 10dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784935 r1", "GSM8784935", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122374.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 411821124.0, 8074924.0, "GSM8784935 r2", "0:51", "A:106698116;C:97949900;G:99548706;T:107586633;N:37769", 51, null, null, null, 106698116, 97949900, 99548706, 107586633, 37769, "SRX27626968", "SRS24034237", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34821, "SRR32289943", "SRX27626967", "SRS24034233", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 4", "GSM8784934", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784934", "GSM8784934: FAC sorted RGCs at 10dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784934 r1", "GSM8784934", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121319.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 320645415.0, 6287165.0, "GSM8784934 r1", "0:51", "A:83667318;C:75715675;G:77201096;T:84031268;N:30058", 51, null, null, null, 83667318, 75715675, 77201096, 84031268, 30058, "SRX27626967", "SRS24034233", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34822, "SRR32289944", "SRX27626967", "SRS24034233", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 4", "GSM8784934", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784934", "GSM8784934: FAC sorted RGCs at 10dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784934 r1", "GSM8784934", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121319.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 326655969.0, 6405019.0, "GSM8784934 r2", "0:51", "A:85253239;C:77113202;G:78588132;T:85671193;N:30203", 51, null, null, null, 85253239, 77113202, 78588132, 85671193, 30203, "SRX27626967", "SRS24034233", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34823, "SRR32289945", "SRX27626966", "SRS24034235", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 3", "GSM8784933", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784933", "GSM8784933: FAC sorted RGCs at 10dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784933 r1", "GSM8784933", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121318.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 383772552.0, 7524952.0, "GSM8784933 r1", "0:51", "A:100725108;C:90120177;G:92143732;T:100747122;N:36413", 51, null, null, null, 100725108, 90120177, 92143732, 100747122, 36413, "SRX27626966", "SRS24034235", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34824, "SRR32289946", "SRX27626966", "SRS24034235", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 3", "GSM8784933", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784933", "GSM8784933: FAC sorted RGCs at 10dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784933 r1", "GSM8784933", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121318.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 392096007.0, 7688157.0, "GSM8784933 r2", "0:51", "A:102939588;C:92028680;G:94033209;T:103058157;N:36373", 51, null, null, null, 102939588, 92028680, 94033209, 103058157, 36373, "SRX27626966", "SRS24034235", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34825, "SRR32289947", "SRX27626965", "SRS24034238", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 2", "GSM8784932", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784932", "GSM8784932: FAC sorted RGCs at 10dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784932 r1", "GSM8784932", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121317.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 396699216.0, 7778416.0, "GSM8784932 r1", "0:51", "A:103913407;C:93411140;G:95295922;T:104041015;N:37732", 51, null, null, null, 103913407, 93411140, 95295922, 104041015, 37732, "SRX27626965", "SRS24034238", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34826, "SRR32289948", "SRX27626965", "SRS24034238", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 2", "GSM8784932", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784932", "GSM8784932: FAC sorted RGCs at 10dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784932 r1", "GSM8784932", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121317.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 407433696.0, 7988896.0, "GSM8784932 r2", "0:51", "A:106734482;C:95879695;G:97797451;T:106984011;N:38057", 51, null, null, null, 106734482, 95879695, 97797451, 106984011, 38057, "SRX27626965", "SRS24034238", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34827, "SRR32289949", "SRX27626964", "SRS24034232", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 1", "GSM8784931", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784931", "GSM8784931: FAC sorted RGCs at 10dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784931 r1", "GSM8784931", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121316.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 357420852.0, 7008252.0, "GSM8784931 r1", "0:51", "A:100707954;C:76739501;G:77474055;T:102464781;N:34561", 51, null, null, null, 100707954, 76739501, 77474055, 102464781, 34561, "SRX27626964", "SRS24034232", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34828, "SRR32289950", "SRX27626964", "SRS24034232", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 10dpi biological replicate 1", "GSM8784931", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 10dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784931", "GSM8784931: FAC sorted RGCs at 10dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784931 r1", "GSM8784931", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121316.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 369272181.0, 7240631.0, "GSM8784931 r2", "0:51", "A:104043292;C:79231475;G:79962686;T:105999425;N:35303", 51, null, null, null, 104043292, 79231475, 79962686, 105999425, 35303, "SRX27626964", "SRS24034232", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34829, "SRR32289951", "SRX27626963", "SRS24034234", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 5", "GSM8784930", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784930", "GSM8784930: FAC sorted RGCs at 6dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784930 r1", "GSM8784930", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122373.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 383701101.0, 7523551.0, "GSM8784930 r1", "0:51", "A:101986811;C:88337442;G:89758931;T:103581971;N:35946", 51, null, null, null, 101986811, 88337442, 89758931, 103581971, 35946, "SRX27626963", "SRS24034234", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34830, "SRR32289952", "SRX27626963", "SRS24034234", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 5", "GSM8784930", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784930", "GSM8784930: FAC sorted RGCs at 6dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784930 r1", "GSM8784930", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122373.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 394847355.0, 7742105.0, "GSM8784930 r2", "0:51", "A:104937168;C:90871625;G:92274823;T:106726849;N:36890", 51, null, null, null, 104937168, 90871625, 92274823, 106726849, 36890, "SRX27626963", "SRS24034234", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34831, "SRR32289953", "SRX27626962", "SRS24034230", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 4", "GSM8784929", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784929", "GSM8784929: FAC sorted RGCs at 6dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784929 r1", "GSM8784929", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121313.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 397357269.0, 7791319.0, "GSM8784929 r1", "0:51", "A:102962865;C:94382913;G:96076584;T:103896489;N:38418", 51, null, null, null, 102962865, 94382913, 96076584, 103896489, 38418, "SRX27626962", "SRS24034230", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34832, "SRR32289954", "SRX27626962", "SRS24034230", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 4", "GSM8784929", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784929", "GSM8784929: FAC sorted RGCs at 6dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784929 r1", "GSM8784929", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121313.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 407641317.0, 7992967.0, "GSM8784929 r2", "0:51", "A:105635966;C:96802890;G:98458161;T:106705494;N:38806", 51, null, null, null, 105635966, 96802890, 98458161, 106705494, 38806, "SRX27626962", "SRS24034230", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34833, "SRR32289955", "SRX27626961", "SRS24034228", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 3", "GSM8784928", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784928", "GSM8784928: FAC sorted RGCs at 6dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784928 r1", "GSM8784928", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121312.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 375101328.0, 7354928.0, "GSM8784928 r1", "0:51", "A:98240052;C:88061621;G:89879822;T:98883790;N:36043", 51, null, null, null, 98240052, 88061621, 89879822, 98883790, 36043, "SRX27626961", "SRS24034228", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34834, "SRR32289956", "SRX27626961", "SRS24034228", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 3", "GSM8784928", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784928", "GSM8784928: FAC sorted RGCs at 6dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784928 r1", "GSM8784928", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121312.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 382030290.0, 7490790.0, "GSM8784928 r2", "0:51", "A:100050525;C:89650775;G:91497813;T:100795370;N:35807", 51, null, null, null, 100050525, 89650775, 91497813, 100795370, 35807, "SRX27626961", "SRS24034228", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34835, "SRR32289957", "SRX27626960", "SRS24034231", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 2", "GSM8784927", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784927", "GSM8784927: FAC sorted RGCs at 6dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784927 r1", "GSM8784927", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121311.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 359457486.0, 7048186.0, "GSM8784927 r1", "0:51", "A:94049871;C:84581283;G:86196196;T:94595815;N:34321", 51, null, null, null, 94049871, 84581283, 86196196, 94595815, 34321, "SRX27626960", "SRS24034231", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34836, "SRR32289958", "SRX27626960", "SRS24034231", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 2", "GSM8784927", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784927", "GSM8784927: FAC sorted RGCs at 6dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784927 r1", "GSM8784927", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121311.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 368062155.0, 7216905.0, "GSM8784927 r2", "0:51", "A:96303888;C:86587511;G:88207050;T:96929121;N:34585", 51, null, null, null, 96303888, 86587511, 88207050, 96929121, 34585, "SRX27626960", "SRS24034231", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34837, "SRR32289959", "SRX27626959", "SRS24034229", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 1", "GSM8784926", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784926", "GSM8784926: FAC sorted RGCs at 6dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784926 r1", "GSM8784926", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121310.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 308100945.0, 6041195.0, "GSM8784926 r1", "0:51", "A:86887196;C:64730880;G:65880791;T:90571586;N:30492", 51, null, null, null, 86887196, 64730880, 65880791, 90571586, 30492, "SRX27626959", "SRS24034229", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34838, "SRR32289960", "SRX27626959", "SRS24034229", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 6dpi biological replicate 1", "GSM8784926", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 6dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784926", "GSM8784926: FAC sorted RGCs at 6dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784926 r1", "GSM8784926", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121310.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 318837465.0, 6251715.0, "GSM8784926 r2", "0:51", "A:89949594;C:66936819;G:68101167;T:93818871;N:31014", 51, null, null, null, 89949594, 66936819, 68101167, 93818871, 31014, "SRX27626959", "SRS24034229", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34839, "SRR32289961", "SRX27626958", "SRS24034226", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 6", "GSM8784925", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784925", "GSM8784925: FAC sorted RGCs at 3dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784925 r1", "GSM8784925", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122372.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 330289260.0, 6476260.0, "GSM8784925 r1", "0:51", "A:86654778;C:77586975;G:79218167;T:86799619;N:29721", 51, null, null, null, 86654778, 77586975, 79218167, 86799619, 29721, "SRX27626958", "SRS24034226", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34840, "SRR32289962", "SRX27626958", "SRS24034226", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 6", "GSM8784925", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784925", "GSM8784925: FAC sorted RGCs at 3dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784925 r1", "GSM8784925", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122372.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 337840524.0, 6624324.0, "GSM8784925 r2", "0:51", "A:88622074;C:79353340;G:80979292;T:88855871;N:29947", 51, null, null, null, 88622074, 79353340, 80979292, 88855871, 29947, "SRX27626958", "SRS24034226", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34841, "SRR32289963", "SRX27626957", "SRS24034227", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 5", "GSM8784924", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784924", "GSM8784924: FAC sorted RGCs at 3dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784924 r1", "GSM8784924", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122371.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 368108820.0, 7217820.0, "GSM8784924 r1", "0:51", "A:98464174;C:82788417;G:84631916;T:102188956;N:35357", 51, null, null, null, 98464174, 82788417, 84631916, 102188956, 35357, "SRX27626957", "SRS24034227", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34842, "SRR32289964", "SRX27626957", "SRS24034227", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 5", "GSM8784924", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784924", "GSM8784924: FAC sorted RGCs at 3dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784924 r1", "GSM8784924", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122371.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 378268275.0, 7417025.0, "GSM8784924 r2", "0:51", "A:101126361;C:85046503;G:86891002;T:105168387;N:36022", 51, null, null, null, 101126361, 85046503, 86891002, 105168387, 36022, "SRX27626957", "SRS24034227", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34843, "SRR32289965", "SRX27626956", "SRS24034225", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 4", "GSM8784923", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784923", "GSM8784923: FAC sorted RGCs at 3dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784923 r1", "GSM8784923", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121307.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 286509432.0, 5617832.0, "GSM8784923 r1", "0:51", "A:73208995;C:69469968;G:70853373;T:72949032;N:28064", 51, null, null, null, 73208995, 69469968, 70853373, 72949032, 28064, "SRX27626956", "SRS24034225", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34844, "SRR32289966", "SRX27626956", "SRS24034225", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 4", "GSM8784923", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784923", "GSM8784923: FAC sorted RGCs at 3dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784923 r1", "GSM8784923", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121307.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 293974863.0, 5764213.0, "GSM8784923 r2", "0:51", "A:75131024;C:71285864;G:72635665;T:74893973;N:28337", 51, null, null, null, 75131024, 71285864, 72635665, 74893973, 28337, "SRX27626956", "SRS24034225", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34845, "SRR32289967", "SRX27626955", "SRS24034223", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 3", "GSM8784922", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784922", "GSM8784922: FAC sorted RGCs at 3dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784922 r1", "GSM8784922", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121306.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 358789233.0, 7035083.0, "GSM8784922 r1", "0:51", "A:93438522;C:84870850;G:86529862;T:93916370;N:33629", 51, null, null, null, 93438522, 84870850, 86529862, 93916370, 33629, "SRX27626955", "SRS24034223", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34846, "SRR32289968", "SRX27626955", "SRS24034223", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 3", "GSM8784922", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784922", "GSM8784922: FAC sorted RGCs at 3dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784922 r1", "GSM8784922", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121306.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 365025258.0, 7157358.0, "GSM8784922 r2", "0:51", "A:95070256;C:86325431;G:87987533;T:95608156;N:33882", 51, null, null, null, 95070256, 86325431, 87987533, 95608156, 33882, "SRX27626955", "SRS24034223", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34847, "SRR32289969", "SRX27626954", "SRS24034224", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 2", "GSM8784921", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784921", "GSM8784921: FAC sorted RGCs at 3dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784921 r1", "GSM8784921", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121305.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 391506702.0, 7676602.0, "GSM8784921 r1", "0:51", "A:101960329;C:92790371;G:94697001;T:102022049;N:36952", 51, null, null, null, 101960329, 92790371, 94697001, 102022049, 36952, "SRX27626954", "SRS24034224", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34848, "SRR32289970", "SRX27626954", "SRS24034224", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 2", "GSM8784921", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784921", "GSM8784921: FAC sorted RGCs at 3dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784921 r1", "GSM8784921", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121305.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 401026872.0, 7863272.0, "GSM8784921 r2", "0:51", "A:104424027;C:95011852;G:96926223;T:104627136;N:37634", 51, null, null, null, 104424027, 95011852, 96926223, 104627136, 37634, "SRX27626954", "SRS24034224", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34849, "SRR32289971", "SRX27626953", "SRS24034222", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 1", "GSM8784920", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784920", "GSM8784920: FAC sorted RGCs at 3dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784920 r1", "GSM8784920", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121304.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 339021174.0, 6647474.0, "GSM8784920 r1", "0:51", "A:93114431;C:74857016;G:76108927;T:94907017;N:33783", 51, null, null, null, 93114431, 74857016, 76108927, 94907017, 33783, "SRX27626953", "SRS24034222", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34850, "SRR32289972", "SRX27626953", "SRS24034222", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 3dpi biological replicate 1", "GSM8784920", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 3dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784920", "GSM8784920: FAC sorted RGCs at 3dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784920 r1", "GSM8784920", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121304.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 353383896.0, 6929096.0, "GSM8784920 r2", "0:51", "A:97070049;C:77993850;G:79259723;T:99025646;N:34628", 51, null, null, null, 97070049, 77993850, 79259723, 99025646, 34628, "SRX27626953", "SRS24034222", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34851, "SRR32289973", "SRX27626952", "SRS24034220", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 7", "GSM8784919", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 7", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784919", "GSM8784919: FAC sorted RGCs at 1dpi biological replicate 7; Danio rerio; RNA Seq", "GSM8784919 r1", "GSM8784919", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122370.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 394021869.0, 7725919.0, "GSM8784919 r1", "0:51", "A:104916639;C:90350948;G:92325077;T:106392449;N:36756", 51, null, null, null, 104916639, 90350948, 92325077, 106392449, 36756, "SRX27626952", "SRS24034220", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34852, "SRR32289974", "SRX27626952", "SRS24034220", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 7", "GSM8784919", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 7", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784919", "GSM8784919: FAC sorted RGCs at 1dpi biological replicate 7; Danio rerio; RNA Seq", "GSM8784919 r1", "GSM8784919", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122370.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 402835893.0, 7898743.0, "GSM8784919 r2", "0:51", "A:107284622;C:92356101;G:94308190;T:108850087;N:36893", 51, null, null, null, 107284622, 92356101, 94308190, 108850087, 36893, "SRX27626952", "SRS24034220", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34853, "SRR32289975", "SRX27626951", "SRS24034221", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 6", "GSM8784918", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784918", "GSM8784918: FAC sorted RGCs at 1dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784918 r1", "GSM8784918", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122368.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 331394226.0, 6497926.0, "GSM8784918 r1", "0:51", "A:92193678;C:70679080;G:72151366;T:96339266;N:30836", 51, null, null, null, 92193678, 70679080, 72151366, 96339266, 30836, "SRX27626951", "SRS24034221", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34854, "SRR32289976", "SRX27626951", "SRS24034221", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 6", "GSM8784918", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784918", "GSM8784918: FAC sorted RGCs at 1dpi biological replicate 6; Danio rerio; RNA Seq", "GSM8784918 r1", "GSM8784918", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122368.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 341222844.0, 6690644.0, "GSM8784918 r2", "0:51", "A:94891966;C:72728396;G:74206983;T:99364490;N:31009", 51, null, null, null, 94891966, 72728396, 74206983, 99364490, 31009, "SRX27626951", "SRS24034221", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34855, "SRR32289977", "SRX27626950", "SRS24034218", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 5", "GSM8784917", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784917", "GSM8784917: FAC sorted RGCs at 1dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784917 r1", "GSM8784917", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122367.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 318762036.0, 6250236.0, "GSM8784917 r1", "0:51", "A:88455336;C:66904117;G:68494022;T:94878199;N:30362", 51, null, null, null, 88455336, 66904117, 68494022, 94878199, 30362, "SRX27626950", "SRS24034218", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34856, "SRR32289978", "SRX27626950", "SRS24034218", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 5", "GSM8784917", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784917", "GSM8784917: FAC sorted RGCs at 1dpi biological replicate 5; Danio rerio; RNA Seq", "GSM8784917 r1", "GSM8784917", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC122367.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 327003024.0, 6411824.0, "GSM8784917 r2", "0:51", "A:90712200;C:68607337;G:70232257;T:97420994;N:30236", 51, null, null, null, 90712200, 68607337, 70232257, 97420994, 30236, "SRX27626950", "SRS24034218", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34857, "SRR32289979", "SRX27626949", "SRS24034219", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 4", "GSM8784916", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784916", "GSM8784916: FAC sorted RGCs at 1dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784916 r1", "GSM8784916", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121301.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 387759885.0, 7603135.0, "GSM8784916 r1", "0:51", "A:102783169;C:88945135;G:91353292;T:104641521;N:36768", 51, null, null, null, 102783169, 88945135, 91353292, 104641521, 36768, "SRX27626949", "SRS24034219", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34858, "SRR32289980", "SRX27626949", "SRS24034219", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 4", "GSM8784916", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784916", "GSM8784916: FAC sorted RGCs at 1dpi biological replicate 4; Danio rerio; RNA Seq", "GSM8784916 r1", "GSM8784916", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121301.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 397404189.0, 7792239.0, "GSM8784916 r2", "0:51", "A:105304214;C:91200661;G:93584392;T:107277465;N:37457", 51, null, null, null, 105304214, 91200661, 93584392, 107277465, 37457, "SRX27626949", "SRS24034219", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34859, "SRR32289981", "SRX27626948", "SRS24034216", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 3", "GSM8784915", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784915", "GSM8784915: FAC sorted RGCs at 1dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784915 r1", "GSM8784915", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121300.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 428248785.0, 8397035.0, "GSM8784915 r1", "0:51", "A:112023158;C:100515159;G:102620937;T:113048752;N:40779", 51, null, null, null, 112023158, 100515159, 102620937, 113048752, 40779, "SRX27626948", "SRS24034216", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34860, "SRR32289982", "SRX27626948", "SRS24034216", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 3", "GSM8784915", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784915", "GSM8784915: FAC sorted RGCs at 1dpi biological replicate 3; Danio rerio; RNA Seq", "GSM8784915 r1", "GSM8784915", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121300.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 437907063.0, 8586413.0, "GSM8784915 r2", "0:51", "A:114573702;C:102734144;G:104841829;T:115716348;N:41040", 51, null, null, null, 114573702, 102734144, 104841829, 115716348, 41040, "SRX27626948", "SRS24034216", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34861, "SRR32289983", "SRX27626947", "SRS24034217", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 2", "GSM8784914", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784914", "GSM8784914: FAC sorted RGCs at 1dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784914 r1", "GSM8784914", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121299.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 402924888.0, 7900488.0, "GSM8784914 r1", "0:51", "A:107947778;C:91046861;G:93204506;T:110687203;N:38540", 51, null, null, null, 107947778, 91046861, 93204506, 110687203, 38540, "SRX27626947", "SRS24034217", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34862, "SRR32289984", "SRX27626947", "SRS24034217", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 2", "GSM8784914", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 2", "GSM8784914", "GSM8784914: FAC sorted RGCs at 1dpi biological replicate 2; Danio rerio; RNA Seq", "GSM8784914 r1", "GSM8784914", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121299.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 413979393.0, 8117243.0, "GSM8784914 r2", "0:51", "A:110924319;C:93515017;G:95646686;T:113854113;N:39258", 51, null, null, null, 110924319, 93515017, 95646686, 113854113, 39258, "SRX27626947", "SRS24034217", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34863, "SRR32289985", "SRX27626946", "SRS24034215", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 1", "GSM8784913", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784913", "GSM8784913: FAC sorted RGCs at 1dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784913 r1", "GSM8784913", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121298.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 360875541.0, 7075991.0, "GSM8784913 r1", "0:51", "A:102005383;C:74767685;G:76535877;T:107531326;N:35270", 51, null, null, null, 102005383, 74767685, 76535877, 107531326, 35270, "SRX27626946", "SRS24034215", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34864, "SRR32289986", "SRX27626946", "SRS24034215", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at 1dpi biological replicate 1", "GSM8784913", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at 1dpi biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:optic nerve crush|batch:RNA extraction batch 1", "GSM8784913", "GSM8784913: FAC sorted RGCs at 1dpi biological replicate 1; Danio rerio; RNA Seq", "GSM8784913 r1", "GSM8784913", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121298.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 372258078.0, 7299178.0, "GSM8784913 r2", "0:51", "A:105213098;C:77053267;G:78863209;T:111092762;N:35742", 51, null, null, null, 105213098, 77053267, 78863209, 111092762, 35742, "SRX27626946", "SRS24034215", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34865, "SRR32289987", "SRX27626945", "SRS24034212", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 6", "GSM8784912", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784912", "GSM8784912: FAC sorted RGCs at naive condition biological replicate 6; Danio rerio; RNA Seq", "GSM8784912 r1", "GSM8784912", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121295.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 304489992.0, 5970392.0, "GSM8784912 r1", "0:51", "A:82425732;C:67909372;G:69611868;T:84514015;N:29005", 51, null, null, null, 82425732, 67909372, 69611868, 84514015, 29005, "SRX27626945", "SRS24034212", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34866, "SRR32289988", "SRX27626945", "SRS24034212", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 6", "GSM8784912", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 6", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784912", "GSM8784912: FAC sorted RGCs at naive condition biological replicate 6; Danio rerio; RNA Seq", "GSM8784912 r1", "GSM8784912", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121295.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 311693232.0, 6111632.0, "GSM8784912 r2", "0:51", "A:84387116;C:69487428;G:71190724;T:86598688;N:29276", 51, null, null, null, 84387116, 69487428, 71190724, 86598688, 29276, "SRX27626945", "SRS24034212", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34867, "SRR32289989", "SRX27626944", "SRS24034214", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 5", "GSM8784911", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784911", "GSM8784911: FAC sorted RGCs at naive condition biological replicate 5; Danio rerio; RNA Seq", "GSM8784911 r1", "GSM8784911", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121294.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 371909136.0, 7292336.0, "GSM8784911 r1", "0:51", "A:97343920;C:87664048;G:89481936;T:97384353;N:34879", 51, null, null, null, 97343920, 87664048, 89481936, 97384353, 34879, "SRX27626944", "SRS24034214", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34868, "SRR32289990", "SRX27626944", "SRS24034214", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 5", "GSM8784911", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 5", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784911", "GSM8784911: FAC sorted RGCs at naive condition biological replicate 5; Danio rerio; RNA Seq", "GSM8784911 r1", "GSM8784911", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121294.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 379291284.0, 7437084.0, "GSM8784911 r2", "0:51", "A:99243449;C:89397613;G:91207377;T:99407666;N:35179", 51, null, null, null, 99243449, 89397613, 91207377, 99407666, 35179, "SRX27626944", "SRS24034214", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34869, "SRR32289991", "SRX27626943", "SRS24034213", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 4", "GSM8784910", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784910", "GSM8784910: FAC sorted RGCs at naive condition biological replicate 4; Danio rerio; RNA Seq", "GSM8784910 r1", "GSM8784910", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121293.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 381808797.0, 7486447.0, "GSM8784910 r1", "0:51", "A:102345832;C:86993840;G:89121190;T:103311576;N:36359", 51, null, null, null, 102345832, 86993840, 89121190, 103311576, 36359, "SRX27626943", "SRS24034213", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34870, "SRR32289992", "SRX27626943", "SRS24034213", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 4", "GSM8784910", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 4", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 2", "GSM8784910", "GSM8784910: FAC sorted RGCs at naive condition biological replicate 4; Danio rerio; RNA Seq", "GSM8784910 r1", "GSM8784910", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121293.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 392824899.0, 7702449.0, "GSM8784910 r2", "0:51", "A:105300909;C:89459531;G:91629489;T:106397902;N:37068", 51, null, null, null, 105300909, 89459531, 91629489, 106397902, 37068, "SRX27626943", "SRS24034213", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34871, "SRR32289993", "SRX27626942", "SRS24034211", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 3", "GSM8784909", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784909", "GSM8784909: FAC sorted RGCs at naive condition biological replicate 3; Danio rerio; RNA Seq", "GSM8784909 r1", "GSM8784909", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121292.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 319281267.0, 6260417.0, "GSM8784909 r1", "0:51", "A:89536872;C:68243564;G:69702232;T:91767357;N:31242", 51, null, null, null, 89536872, 68243564, 69702232, 91767357, 31242, "SRX27626942", "SRS24034211", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34872, "SRR32289994", "SRX27626942", "SRS24034211", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 3", "GSM8784909", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 3", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784909", "GSM8784909: FAC sorted RGCs at naive condition biological replicate 3; Danio rerio; RNA Seq", "GSM8784909 r1", "GSM8784909", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121292.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 330633631.0, 6483013.0, "GSM8784909 r2", "0:51.00", "A:92715496;C:70634109;G:72135232;T:95116600;N:32194", 51, null, null, null, 92715496, 70634109, 72135232, 95116600, 32194, "SRX27626942", "SRS24034211", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34873, "SRR32289995", "SRX27626941", "SRS24034208", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 2", "GSM8784908", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784908", "GSM8784908: FAC sorted RGCs at naive condition biological replicate 2; Danio rerio; RNA Seq", "GSM8784908 r1", "GSM8784908", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121291.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 331915752.0, 6508152.0, "GSM8784908 r1", "0:51", "A:93895773;C:69919819;G:71545387;T:96523051;N:31722", 51, null, null, null, 93895773, 69919819, 71545387, 96523051, 31722, "SRX27626941", "SRS24034208", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34874, "SRR32289996", "SRX27626941", "SRS24034208", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 2", "GSM8784908", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 2", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784908", "GSM8784908: FAC sorted RGCs at naive condition biological replicate 2; Danio rerio; RNA Seq", "GSM8784908 r1", "GSM8784908", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121291.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 341705712.0, 6700112.0, "GSM8784908 r2", "0:51", "A:96635678;C:71919743;G:73610334;T:99507718;N:32239", 51, null, null, null, 96635678, 71919743, 73610334, 99507718, 32239, "SRX27626941", "SRS24034208", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34875, "SRR32289997", "SRX27626940", "SRS24034209", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 1", "GSM8784907", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784907", "GSM8784907: FAC sorted RGCs at naive condition biological replicate 1; Danio rerio; RNA Seq", "GSM8784907 r1", "GSM8784907", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121290.220715.HiSeq4000.FCB.lane6.gcap_18_12.R1.fastq.gz", "fastq", 373593207.0, 7325357.0, "GSM8784907 r1", "0:51", "A:104253639;C:80473294;G:82236043;T:106593718;N:36513", 51, null, null, null, 104253639, 80473294, 82236043, 106593718, 36513, "SRX27626940", "SRS24034209", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34876, "SRR32289998", "SRX27626940", "SRS24034209", "SRP562853", "PRJNA1221797", "Successful axonal regeneration is driven by evolutionarily conserved metabolic reprogramming", "GSE289140", "Transcriptome Analysis", "Unlike mammals  zebrafish can regrow xxx post injury and restore circuit function in the central nervous system CNS. Mitochondria have been identified as key players in this process  but how different metabolic pathways work together to sustain regeneration remains unclear. Using RNA sequencing of adult zebrafish retinal ganglion cells RGCs post optic nerve crush injury  we demonstrate that oxidative phosphorylation is downregulated during axonal regrowth. Simultaneously  the thioredoxin antioxidant system is upregulated  likely to limit oxidative damage. Additionally  we observe an integrated upregulation of glycolysis and the pentose phosphate pathway during the initial regrowth phases  possibly to provide energy while supplying NADPH for biosynthesis and antioxidant responses. We show that this metabolic reprogramming is evolutionarily conserved  as it also occurs in the pro regenerative mammalian Pten and Socs3 co deletion model. Inhibiting glycolysis and thioredoxin in zebrafish impairs axonal regrowth  suggesting that targeting these pathways could enhance CNS regeneration in mammals. Overall design: Bulk RNA sequencing of FAC sorted adult Tgisl2b:eGFPzc7Tg zebrafish retinal ganglion cells under the uninjured naive condition and at 1  3  6  10  and 14 days post optic nerve crush injury.", null, null, null, "FAC sorted RGCs at naive condition biological replicate 1", "GSM8784907", null, "tissue:Retinal ganglion cells|cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1|geo loc name:missing|collection date:missing", "FAC sorted RGCs at naive condition biological replicate 1", "Technical replicates of RNA seq samples were merged post sequencing  and adapter sequences were removed using TrimGalore v0.6.7. Quality control was conducted using FastQC v0.11.5. Sequencing reads were aligned to the zebrafish reference genome Ensembl GRCz11 using the STAR aligner. Genes with low expression were filtered out using a custom developed R script. Batch effects were corrected with pyCombat. Assembly: Ensembl GRCz11 Supplementary files format and content: Tab delimited text file includes raw counts for each sample Supplementary files format and content: Filtered and batch effects corrected counts for all samples", "Retinal ganglion cells", null, "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer\u2019s instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "cell type:Retinal ganglion cells|genotype:Tgisl2b:eGFPzc7Tg|treatment:Non injured|batch:RNA extraction batch 1", "GSM8784907", "GSM8784907: FAC sorted RGCs at naive condition biological replicate 1; Danio rerio; RNA Seq", "GSM8784907 r1", "GSM8784907", "1", "RNA was extracted in two separate batches using the Quick RNA Microprep kit according to the manufacturer's instructions. Libraries were prepared using the Smart Seq2 method RGC samples  Nextera XT DNA  Illumina  San Diego  CA  USA", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP562853", null, null, "GC121290.220715.HiSeq4000.FCB.lane7.gcap_18_12.R1.fastq.gz", "fastq", 384155460.0, 7532460.0, "GSM8784907 r2", "0:51", "A:107214095;C:82732789;G:84490928;T:109680699;N:36949", 51, null, null, null, 107214095, 82732789, 84490928, 109680699, 36949, "SRX27626940", "SRS24034209", "SRA2075201", "KU Leuven", "KU Leuven", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "Belgium", "2025-02-10", "Undetermined", "Adult", "Eye", "Sensory System"], [34913, "SRR32335084", "SRX27670022", "SRS24074877", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "crim1 knockdown whole eyes  72 hpf  sample 3", "GSM8794307", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 / |geo loc name:missing|collection date:missing", "crim1 knockdown whole eyes  72 hpf  sample 3", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:crim1 / ", "GSM8794307", "GSM8794307: crim1 knockdown whole eyes  72 hpf  sample 3; Danio rerio; RNA Seq", "GSM8794307 r1", "GSM8794307", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-dpf-3_S15_L002_R1_001.fastq.gz", "fastq", 3331893906.0, 50483241.0, "GSM8794307 r1", "0:66", "A:873905674;C:752237090;G:742087342;T:963442356;N:221444", 66, null, null, null, 873905674, 752237090, 742087342, 963442356, 221444, "SRX27670022", "SRS24074877", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34914, "SRR32335085", "SRX27670021", "SRS24074876", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "crim1 knockdown whole eyes  72 hpf  sample 2", "GSM8794306", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 / |geo loc name:missing|collection date:missing", "crim1 knockdown whole eyes  72 hpf  sample 2", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:crim1 / ", "GSM8794306", "GSM8794306: crim1 knockdown whole eyes  72 hpf  sample 2; Danio rerio; RNA Seq", "GSM8794306 r1", "GSM8794306", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-dpf-2_S11_L002_R1_001.fastq.gz", "fastq", 3491059044.0, 52894834.0, "GSM8794306 r1", "0:66", "A:907771486;C:794245897;G:783248243;T:1005559302;N:234116", 66, null, null, null, 907771486, 794245897, 783248243, 1005559302, 234116, "SRX27670021", "SRS24074876", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34915, "SRR32335086", "SRX27670020", "SRS24074875", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "crim1 knockdown whole eyes  72 hpf  sample 1", "GSM8794305", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 /. |geo loc name:missing|collection date:missing", "crim1 knockdown whole eyes  72 hpf  sample 1", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:crim1 /. ", "GSM8794305", "GSM8794305: crim1 knockdown whole eyes  72 hpf  sample 1; Danio rerio; RNA Seq", "GSM8794305 r1", "GSM8794305", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-dpf-1_S16_L003_R1_001.fastq.gz", "fastq", 2751667380.0, 41691930.0, "GSM8794305 r1", "0:66", "A:714065903;C:618520760;G:607652792;T:811160146;N:267779", 66, null, null, null, 714065903, 618520760, 607652792, 811160146, 267779, "SRX27670020", "SRS24074875", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34916, "SRR32335087", "SRX27670019", "SRS24074874", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "wildtype whole eyes  72 hpf  sample 3", "GSM8794304", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing", "wildtype whole eyes  72 hpf  sample 3", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:Wildtype", "GSM8794304", "GSM8794304: wildtype whole eyes  72 hpf  sample 3; Danio rerio; RNA Seq", "GSM8794304 r1", "GSM8794304", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-ctr-3_S12_L002_R1_001.fastq.gz", "fastq", 2881865976.0, 43664636.0, "GSM8794304 r1", "0:66", "A:759373846;C:644323432;G:637932338;T:840046555;N:189805", 66, null, null, null, 759373846, 644323432, 637932338, 840046555, 189805, "SRX27670019", "SRS24074874", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34917, "SRR32335088", "SRX27670018", "SRS24074873", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "wildtype whole eyes  72 hpf  sample 2", "GSM8794303", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing", "wildtype whole eyes  72 hpf  sample 2", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:Wildtype", "GSM8794303", "GSM8794303: wildtype whole eyes  72 hpf  sample 2; Danio rerio; RNA Seq", "GSM8794303 r1", "GSM8794303", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-ctr-2_S20_L003_R1_001.fastq.gz", "fastq", 2380780182.0, 36072427.0, "GSM8794303 r1", "0:66", "A:617266566;C:538355433;G:528639561;T:696293971;N:224651", 66, null, null, null, 617266566, 538355433, 528639561, 696293971, 224651, "SRX27670018", "SRS24074873", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34918, "SRR32335089", "SRX27670017", "SRS24074872", "SRP563805", "PRJNA1223441", "A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ", "GSE289562", "Transcriptome Analysis", "We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.", null, "pubmed:40114969", null, "wildtype whole eyes  72 hpf  sample 1", "GSM8794302", null, "source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing", "wildtype whole eyes  72 hpf  sample 1", "The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than \u00b10.5 with an adjusted p value \u22640.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM \u22651 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.", "Whole eye", null, "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed \u2018bulk\u2019 RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "tissue:Whole eye|cell type:All|genotype:Wildtype", "GSM8794302", "GSM8794302: wildtype whole eyes  72 hpf  sample 1; Danio rerio; RNA Seq", "GSM8794302 r1", "GSM8794302", "1", "Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation\u00ae RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5\u2032 end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3\u2032 end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP563805", null, null, "crim1-ctr-1_S17_L003_R1_001.fastq.gz", "fastq", 3007758600.0, 45572100.0, "GSM8794302 r1", "0:66", "A:783035033;C:680405728;G:668894784;T:875142229;N:280826", 66, null, null, null, 783035033, 680405728, 668894784, 875142229, 280826, "SRX27670017", "SRS24074872", null, null, "Cincinnati Children's Hospital Medical Center", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "unknown", "bulk", "bulk", "bulk", null, "United States", "2025-02-13", "Larval", "Larval", "Eye", "Sensory System"], [34962, "SRR32588719", "SRX27895232", "SRS24266231", "SRP568323", "PRJNA1232602", "Telomere to telomere genome assemblies for commonly used zebrafish laboratory strains", "PRJNA1232602", "Other", "In this study  Iso Seq was performed on different zebrafish body organs  enabling a comprehensive view of organ specific transcriptomes. High quality PacBio Iso Seq long reads were generated from key zebrafish tissues  including the brain  testis  liver  eye  muscle  ovary  inner ear and kidney  to identify novel isoforms  tissue specific transcripts  and alternative splicing events. We also made available the Iso Seq data from embryos from different time points  hpf  0  6  12  and 24. This dataset enhances gene annotation  improves reference genome annotations  and provides insights into zebrafish organ specific gene expression.", null, null, null, "Iso Seq RNA from Danio rerio", "C2 F2 F EYE", null, "strain:NHGRI2|isolate:Single paired cross|breed:Zebrafish|cultivar:wild type|ecotype:United States|age:9 month|dev stage:adult fish|collection date:2024 06|geo loc name:USA|sex:female|tissue:eye|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "Iso Seq RNA from eye", "C2 F2 F EYE", "C2 F2 F EYE", "The long Read Sequencing libraries was sequenced using PacBio Sequel II", null, null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "PACBIO_SMRT", "Sequel II", null, "SRP568323", null, null, "m64467e_240703_141215.skera.flnc.fastq.gz", "fastq", 36628626441.0, 15348616.0, "m64467e 240703 141215.skera.flnc.fastq.gz", "0:2386.44", "A:10655476547;C:7616418469;G:7889834035;T:10466897390;N:0", 2386, null, null, null, 10655476547, 7616418469, 7889834035, 10466897390, 0, "SRX27895232", "SRS24266231", "SRA2089085", "National Human Genome Research Institute|Translational and Functional Genomics Branch", "National Human Genome Research Institute National Human Genome Research Institute", null, null, null, null, null, null, null, null, null, null, null, "T", null, "long read", "pacbio", "pacbio_modern", "unknown", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United States", "2025-03-06", "Adult", "Adult", "Eye", "Sensory System"], [34972, "SRR32588729", "SRX27895222", "SRS24266219", "SRP568323", "PRJNA1232602", "Telomere to telomere genome assemblies for commonly used zebrafish laboratory strains", "PRJNA1232602", "Other", "In this study  Iso Seq was performed on different zebrafish body organs  enabling a comprehensive view of organ specific transcriptomes. High quality PacBio Iso Seq long reads were generated from key zebrafish tissues  including the brain  testis  liver  eye  muscle  ovary  inner ear and kidney  to identify novel isoforms  tissue specific transcripts  and alternative splicing events. We also made available the Iso Seq data from embryos from different time points  hpf  0  6  12  and 24. This dataset enhances gene annotation  improves reference genome annotations  and provides insights into zebrafish organ specific gene expression.", null, null, null, "Iso Seq RNA from Danio rerio", "C2 F2 M EYE", null, "strain:NHGRI2|isolate:Single paired cross|breed:Zebrafish|cultivar:wild type|ecotype:United States|age:9 month|dev stage:adult fish|collection date:2024 06|geo loc name:USA|sex:male|tissue:eye|BioSampleModel:Model organism or animal", null, null, null, null, null, null, null, null, "Iso Seq RNA from eye", "C2 F2 M EYE", "C2 F2 M EYE", "The long Read Sequencing libraries was sequenced using PacBio Sequel II", null, null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "PACBIO_SMRT", "Sequel II", null, "SRP568323", null, null, "m84270_240904_172441_s3.skera.flnc.fastq.gz", "fastq", 80921799737.0, 39038804.0, "m84270 240904 172441 s3.skera.flnc.fastq.gz", "0:2072.86", "A:23445079372;C:17292916271;G:17758249005;T:22425555089;N:0", 2072, null, null, null, 23445079372, 17292916271, 17758249005, 22425555089, 0, "SRX27895222", "SRS24266219", "SRA2089085", "National Human Genome Research Institute|Translational and Functional Genomics Branch", "National Human Genome Research Institute National Human Genome Research Institute", null, null, null, null, null, null, null, null, null, null, null, "T", null, "long read", "pacbio", "pacbio_modern", "unknown", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United States", "2025-03-06", "Adult", "Adult", "Eye", "Sensory System"], [36699, "SRR835180", "SRX271976", "SRS416272", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 143h 31 436", "GSM1129638", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 143h 31 436", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129638", "GSM1129638: rpeh 143h 31 436; Danio rerio; RNA Seq", "GSM1129638 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129638", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_143h_31_436_ATCT.fq.bz2", "fastq", 233585940.0, 5561570.0, "GSM1129638 r1", "0:42", "A:55598949;C:61313735;G:51248287;T:65240290;N:184679", 42, null, null, null, 55598949, 61313735, 51248287, 65240290, 184679, "SRX271976", "SRS416272", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.81459, null, 0.17202, null, 0.85642, null, 0.45987, null, 42, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"], [36700, "SRR835179", "SRX271975", "SRS416270", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 143h 31 351", "GSM1129637", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 143h 31 351", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129637", "GSM1129637: rpeh 143h 31 351; Danio rerio; RNA Seq", "GSM1129637 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129637", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_143h_31_351_ATCT.fq.bz2", "fastq", 130461444.0, 3623929.0, "GSM1129637 r1", "0:36", "A:30448136;C:34618233;G:27756164;T:37637830;N:1081", 36, null, null, null, 30448136, 34618233, 27756164, 37637830, 1081, "SRX271975", "SRS416270", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.80013, null, 0.18213, null, 0.85673, null, 0.46503, null, 36, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"], [36701, "SRR835178", "SRX271974", "SRS416271", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 86h 33 436", "GSM1129636", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 86h 33 436", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129636", "GSM1129636: rpeh 86h 33 436; Danio rerio; RNA Seq", "GSM1129636 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129636", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_86h_33_436_CTTT.fq.bz2", "fastq", 375701970.0, 8945285.0, "GSM1129636 r1", "0:42", "A:79041659;C:105394908;G:87165145;T:103778792;N:321466", 42, null, null, null, 79041659, 105394908, 87165145, 103778792, 321466, "SRX271974", "SRS416271", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.69127, null, 0.03078, null, 0.88016, null, 0.45532, null, 42, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"], [36702, "SRR835177", "SRX271973", "SRS416269", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 86h 33 351", "GSM1129635", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 86h 33 351", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129635", "GSM1129635: rpeh 86h 33 351; Danio rerio; RNA Seq", "GSM1129635 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129635", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_86h_33_351_CTTT.fq.bz2", "fastq", 94727196.0, 2631311.0, "GSM1129635 r1", "0:36", "A:19052917;C:27063976;G:21468497;T:27141011;N:795", 36, null, null, null, 19052917, 27063976, 21468497, 27141011, 795, "SRX271973", "SRS416269", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.63527, null, 0.03789, null, 0.88118, null, 0.42915, null, 36, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"], [36703, "SRR835176", "SRX271972", "SRS416268", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 86h 32 436", "GSM1129634", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 86h 32 436", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129634", "GSM1129634: rpeh 86h 32 436; Danio rerio; RNA Seq", "GSM1129634 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129634", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_86h_32_436_ACGT.fq.bz2", "fastq", 455528976.0, 10845928.0, "GSM1129634 r1", "0:42", "A:104114782;C:122028793;G:112429635;T:116603169;N:352597", 42, null, null, null, 104114782, 122028793, 112429635, 116603169, 352597, "SRX271972", "SRS416268", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.80425, null, 0.07901, null, 0.83999, null, 0.53561, null, 42, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"], [36704, "SRR835175", "SRX271971", "SRS416267", "SRP021517", "PRJNA198908", "Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin", "GSE46387", "Transcriptome Analysis", "In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing", null, "pubmed:23874447", null, "rpeh 86h 32 351", "GSM1129633", null, "tissue:retinal pigmented epithelium|hpf", "rpeh 86h 32 351", "Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library", "retinal pigmented epithelium", null, "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", null, "hpf", "GSM1129633", "GSM1129633: rpeh 86h 32 351; Danio rerio; RNA Seq", "GSM1129633 1", null, "1", "This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25\u00b0C in mitfavc7.  When held at 32\u00b0C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25\u00b0C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37\u00b0C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37\u00b0C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4\u00b0C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4\u00b0C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400\u00b5L of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4\u00b0C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4\u00b0C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100\u00b5L containing the pigment cell pellet.  Cells were resuspended with 50\u00b5L of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500\u00b5L of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500\u00b5L cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1\u00b5m or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads\u00ae mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3\u2019 end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.", "GEO Accession:GSM1129633", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "SRP021517", null, null, "rpeh_86h_32_351_ACGT.fq.bz2", "fastq", 124319592.0, 3453322.0, "GSM1129633 r1", "0:36", "A:28020637;C:33641464;G:30211380;T:32445126;N:985", 36, null, null, null, 28020637, 33641464, 30211380, 32445126, 985, "SRX271971", "SRS416267", "SRA074390", "GEO", "Rob Mitra, Genetics, Washington University", 1, 0.80002, null, 0.09087, null, 0.83755, null, 0.53605, null, 36, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "United States", "2013-04-25", "Larval", "Larval", "Eye", "Sensory System"]], "truncated": false, "filtered_table_rows_count": 486, "expanded_columns": [], "expandable_columns": [], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": [], "units": {}, "query": {"sql": "select rowid, [run.accession], [experiment.accession], [sample.accession], [study.accession], bioproject, [study.title], [study.alias], [study.type], [study.abstract], [study.attributes], [study.PMIDs], [sample.description], [sample.title], [sample.alias], [sample.centername], [sample.attributes], [GEOsample.title], [GEOsample.dataprocessing], [GEOsample.source], [GEOsample.treatmentprotocol], [GEOsample.extractprotocol], [GEOsample.growthprotocol], [GEOsample.characteristics], [GEOsample.accession], [experiment.title], [experiment.alias], [experiment.library_name], [experiment.design_description], [experiment.library_construction_protocol], [experiment.attributes], [experiment.library_strategy], [experiment.library_source], [experiment.library_selection], [experiment.library_layout], [experiment.platform], [experiment.instrument_model], [experiment.spot_descriptor], [experiment.study_ref], [run.title], [run.attributes], [run.filename], [run.semantic_name], [run.total_bases], [run.total_spots], [run.alias], [run.read_lengths], [run.base_counts], [run.r1_length], [run.r2_length], [run.r3_length], [run.r4_length], [run.Acount], [run.Ccount], [run.Gcount], [run.Tcount], [run.Ncount], [run.experiment], [run.pool_member], [submission.accession], [submission.srasource], [submission.bioprojectsource], [seqdetective.n_mates], [seqdetective.mapping_rate.mate1], [seqdetective.mapping_rate.mate2], [seqdetective.nofeature_rate.mate1], [seqdetective.nofeature_rate.mate2], [seqdetective.sparsity.mate1], [seqdetective.sparsity.mate2], [seqdetective.pos_strand_rate.mate1], [seqdetective.pos_strand_rate.mate2], [seqdetective.readlen.mate1], [seqdetective.readlen.mate2], [seqdetective.judgement.mate1], [seqdetective.judgement.mate2], [seqdetective.judgement.reason], platform_family, instrument_generation, read_bias, selection_class, prep_kit, sc_or_bulk, tech_class, technology, tech_variant, [submission.bioprojectsource.country], earliest_date, devstage_curation, devstage_curation_coarse, tissue_curation, tissue_curation_coarse from run_metadata where \"experiment.library_layout\" = :p0 and \"experiment.library_selection\" = :p1 and \"tissue_curation\" = :p2 order by rowid limit 101", "params": {"p0": "SINGLE", "p1": "cDNA", "p2": "Eye"}}, "facet_results": {"experiment.library_strategy": {"name": "experiment.library_strategy", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye", "results": [{"value": "RNA-Seq", "label": "RNA-Seq", "count": 486, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&experiment.library_strategy=RNA-Seq", "selected": false}], "truncated": false}, "experiment.library_source": {"name": "experiment.library_source", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye", "results": [{"value": "TRANSCRIPTOMIC", "label": "TRANSCRIPTOMIC", "count": 470, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&experiment.library_source=TRANSCRIPTOMIC", "selected": false}, {"value": "TRANSCRIPTOMIC SINGLE CELL", "label": "TRANSCRIPTOMIC SINGLE CELL", "count": 16, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&experiment.library_source=TRANSCRIPTOMIC+SINGLE+CELL", "selected": false}], "truncated": false}, "experiment.library_selection": {"name": "experiment.library_selection", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye", "results": [{"value": "cDNA", "label": "cDNA", "count": 486, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&tissue_curation=Eye", "selected": true}], "truncated": false}, "experiment.library_layout": {"name": "experiment.library_layout", "type": "column", "hideable": false, "toggle_url": "/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye", "results": [{"value": "SINGLE", "label": "SINGLE", "count": 486, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_selection=cDNA&tissue_curation=Eye", "selected": true}], "truncated": false}, "experiment.platform": {"name": "experiment.platform", "type": "column", "hideable": false, "toggle_url": 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"toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&devstage_curation_coarse=Adult", "selected": false}, {"value": "Embryo", "label": "Embryo", "count": 84, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&devstage_curation_coarse=Embryo", "selected": false}, {"value": "Larval", "label": "Larval", "count": 84, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&devstage_curation_coarse=Larval", "selected": false}, {"value": "Juvenile", "label": "Juvenile", "count": 47, "toggle_url": "http://metadata.rnaquarium.org/metadata/run_metadata.json?experiment.library_layout=SINGLE&experiment.library_selection=cDNA&tissue_curation=Eye&devstage_curation_coarse=Juvenile", 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