{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.platform = \"ILLUMINA\", technology = \"10x\" and tissue_curation_coarse = \"Nervous System\"", "rows": [[10176, "ERR5858457", "ERX5504346", "ERS6343450", "ERP128749", "PRJEB44676", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "E-MTAB-10390", "Transcriptome Analysis", "To analyse lesion induced gene regulation in progenitor cells at single cell resolution  we performed single cell RNAseq on FACS isolated her4.3:GFP progenitor cells from the spinal cord at 24 hours post lesion hpl post spinal injury at 3 dpf dpf  compared to age matched uninjured animals.", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", null, "Protocols: Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Naive", "SAMEA8658904", "University Of Edinburgh", "ENA first public:2021 05 24|ENA last update:2021 05 24|External Id:SAMEA8658904|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2021 05 24T00:14:32Z|INSDC last update:2021 05 24T00:14:32Z|INSDC status:public|Submitter Id:E MTAB 10390:Naive|age:4|broker name:ArrayExpress|cell type:ependymo radial glial cell|common name:zebrafish|developmental stage:larval day 4|immunophenotype:Her4.3+ positive|organism part:spinal cord|sample name:E MTAB 10390:Naive|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesi1d and unlesi1d zebrafish larvae spinal cord", "E MTAB 10390:Naive p", "Naive p", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines  Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Experimental Factor: injury:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "Oligo-dT", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP128749", "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", "Naive.bam", "bam", 44972162730.0, 499690697.0, "E MTAB 10390:Naive", "0:90", "A:13658826375;C:8733304343;G:9348137569;T:13228064343;N:3830100", 90, null, null, null, 13658826375, 8733304343, 9348137569, 13228064343, 3830100, "ERX5504346", "ERS6343450", "ERA4142789", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 1, 0.89428, null, 0.32693, null, 0.75276, null, 0.5314, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2021-05-24", "Larval", "Larval", "Spinal Cord", "Nervous System"], [10177, "ERR5858456", "ERX5504345", "ERS6343449", "ERP128749", "PRJEB44676", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "E-MTAB-10390", "Transcriptome Analysis", "To analyse lesion induced gene regulation in progenitor cells at single cell resolution  we performed single cell RNAseq on FACS isolated her4.3:GFP progenitor cells from the spinal cord at 24 hours post lesion hpl post spinal injury at 3 dpf dpf  compared to age matched uninjured animals.", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", null, "Protocols: Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Lesi1d", "SAMEA8658903", "University Of Edinburgh", "ENA first public:2021 05 24|ENA last update:2021 05 24|External Id:SAMEA8658903|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2021 05 24T00:14:32Z|INSDC last update:2021 05 24T00:14:32Z|INSDC status:public|Submitter Id:E MTAB 10390:Lesi1d|age:4|broker name:ArrayExpress|cell type:ependymo radial glial cell|common name:zebrafish|developmental stage:larval day 4|immunophenotype:Her4.3+ positive|injury:spinal injury lesion|organism part:spinal cord|sample name:E MTAB 10390:Lesi1d|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesi1d and unlesi1d zebrafish larvae spinal cord", "E MTAB 10390:Lesioned p", "Lesioned p", "scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "Trunks containing the lesion sites or equivalent site from unlesioned fish are collected and kept in PBS on ice.Incubate the trunks up to 300 in 1 mL of 1X Trypsin EDTA at 37C for 5 7 mins.Stop dissociation by adding FBS to a final concentration of 5%.Centrifuge at 200g for 7 mins.Discard Sups.Resuspend in 500 uL of PBS.Add the cell suspension into a 40 uM cell strainer.Centrifuge at 200g for 7 mins.Resuspend in the buffer for FACS PBS 5% FBS Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines  Following cell dissociation and FAC sorting  samples were processed on the 10X Chromium platform using 10X Single Cell three prime v3 chemistry following the manufacturer's guidelines.", "Experimental Factor: injury:spinal injury lesion", "RNA-Seq", "TRANSCRIPTOMIC", "Oligo-dT", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP128749", "Illumina NovaSeq 6000 paired end sequencing; scRNAseq of her4.3+ cells from lesioned and unlesioned zebrafish larvae spinal cord", "ENA FIRST PUBLIC:2021 05 24|ENA LAST UPDATE:2021 05 24", "Lesioned.bam", "bam", 49902588630.0, 554473207.0, "E MTAB 10390:Lesioned", "0:90", "A:14713351178;C:10191139050;G:10897887675;T:14095967201;N:4243526", 90, null, null, null, 14713351178, 10191139050, 10897887675, 14095967201, 4243526, "ERX5504345", "ERS6343449", "ERA4142789", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 1, 0.91197, null, 0.29137, null, 0.7568, null, 0.56523, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2021-05-24", "Larval", "Larval", "Spinal Cord", "Nervous System"], [11813, "ERR11788863", "ERX11187331", "ERS16221143", "ERP149944", "PRJEB64780", "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "E-MTAB-13223", "Transcriptome Analysis", "To understand the cellular basis of the immune compartment of the zebrafish brain we have established reliable protocols for dissociation and prospective isolation of brain leukocytes  using fluorescent transgenic lines. By combining this approach with single cell RNA sequencing  we have generated a gene expression atlas composed of the distinct immune cells present in the homeostatic brain. These analyses revealed the presence of subpopulations of mononuclear phagocytes and other leukocytes  including cell types that have not been  or have been poorly  characterized so far. Here  we present the characterization of a new mononuclear phagocyte population that represents an important fraction among all brain leukocytes.  Adult brain single cell suspensions were prepared from adult Tgp2ry12:GFP; cd45:DsRed fish n=3 and a total of 14 000 cd45:DsRed+ cells were processed for single cell profiling using the 10x Genomics platform.", "ENA FIRST PUBLIC:2023 12 25|ENA LAST UPDATE:2023 12 25", null, "Protocols: FACS sorting of cd45:Dsred cells. Brains from adult Tgp2ry12:GFP; cd45:DsRed fish n=3  were dissected in 0.9X Dulbecco\u2032s Phosphate Buffered Saline DPBS were triturated and treated with Liberase TM at 33\u00b0C for 30 45 minutes  fully dissociated using a syringe with a 26G needle and washed in 2% fetal bovine serum diluted in 0.9X DPBS. Cell suspensions were centrifuged at 290g 4\u00baC 10 min and filtered through a 40\u00b5m nylon mesh. Just before flow cytometry analysis  using calcein violet to exclude dead cells 1\u00b5M  Thermo Fisher. Flow cytometry acquisition and cell sorting was performed on a FACS ARIA II Becton Dickinson. RNA Illumina", "Sample 1", "SAMEA114235870", "Universite Libre de Bruxelles", "ENA FIRST PUBLIC:2023 12 25T01:15:29Z|ENA LAST UPDATE:2023 12 25T01:15:29Z|External Id:SAMEA114235870|INSDC center name:Universite Libre de Bruxelles|INSDC first public:2023 12 25T01:15:29Z|INSDC last update:2023 12 25T01:15:29Z|INSDC status:public|Submitter Id:E MTAB 13223:Sample 1|age:5|broker name:ArrayExpress|cell type:leukocyte|collection date:not collected|common name:zebrafish|developmental stage:adult|genotype:wild type genotype|geographic location country and/or sea:not collected|individual:pool of 3 fish|isolate:not applicable|organism part:brain|sample name:E MTAB 13223:Sample 1|scientific name:Danio rerio|sex:male and female|strain:AB", null, null, null, null, null, null, null, null, "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "E MTAB 13223:Sample 1 p", "Sample 1 p", "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "FACS sorting of cd45:Dsred cells. Brains from adult Tgp2ry12:GFP; cd45:DsRed fish n=3  were dissected in 0.9X Dulbecco\u2032s Phosphate Buffered Saline DPBS were triturated and treated with Liberase TM at 33\u00b0C for 30 45 minutes  fully dissociated using a syringe with a 26G needle and washed in 2% fetal bovine serum diluted in 0.9X DPBS. Cell suspensions were centrifuged at 290g 4\u00baC 10 min and filtered through a 40\u00b5m nylon mesh. Just before flow cytometry analysis  using calcein violet to exclude dead cells 1\u00b5M  Thermo Fisher. Flow cytometry acquisition and cell sorting was performed on a FACS ARIA II Becton Dickinson.  RNA Illumina", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "PAIRED", "ILLUMINA", "NextSeq 550", null, "ERP149944", "NextSeq 550 paired end sequencing; A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "ENA FIRST PUBLIC:2023 12 25|ENA LAST UPDATE:2023 12 25", "L41805_Track-81843_R1.fastq.gz L41805_Track-81843_R2.fastq.gz", "fastq fastq", 5545444464.0, 66017196.0, "E MTAB 13223:L41805 Track 81843", "0:28 1:56", "A:1576174024;C:1193175858;G:1221776615;T:1551009004;N:3308963", 28, 56, null, null, 1576174024, 1193175858, 1221776615, 1551009004, 3308963, "ERX11187331", "ERS16221143", "ERA26318932", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.00738, 0.90151, 0.00285, 0.20553, 0.99072, 0.82402, 0.4645, 0.53817, 28, 56, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-12-25", "Adult", "Adult", "Brain", "Nervous System"], [11814, "ERR11788864", "ERX11187331", "ERS16221143", "ERP149944", "PRJEB64780", "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "E-MTAB-13223", "Transcriptome Analysis", "To understand the cellular basis of the immune compartment of the zebrafish brain we have established reliable protocols for dissociation and prospective isolation of brain leukocytes  using fluorescent transgenic lines. By combining this approach with single cell RNA sequencing  we have generated a gene expression atlas composed of the distinct immune cells present in the homeostatic brain. These analyses revealed the presence of subpopulations of mononuclear phagocytes and other leukocytes  including cell types that have not been  or have been poorly  characterized so far. Here  we present the characterization of a new mononuclear phagocyte population that represents an important fraction among all brain leukocytes.  Adult brain single cell suspensions were prepared from adult Tgp2ry12:GFP; cd45:DsRed fish n=3 and a total of 14 000 cd45:DsRed+ cells were processed for single cell profiling using the 10x Genomics platform.", "ENA FIRST PUBLIC:2023 12 25|ENA LAST UPDATE:2023 12 25", null, "Protocols: FACS sorting of cd45:Dsred cells. Brains from adult Tgp2ry12:GFP; cd45:DsRed fish n=3  were dissected in 0.9X Dulbecco\u2032s Phosphate Buffered Saline DPBS were triturated and treated with Liberase TM at 33\u00b0C for 30 45 minutes  fully dissociated using a syringe with a 26G needle and washed in 2% fetal bovine serum diluted in 0.9X DPBS. Cell suspensions were centrifuged at 290g 4\u00baC 10 min and filtered through a 40\u00b5m nylon mesh. Just before flow cytometry analysis  using calcein violet to exclude dead cells 1\u00b5M  Thermo Fisher. Flow cytometry acquisition and cell sorting was performed on a FACS ARIA II Becton Dickinson. RNA Illumina", "Sample 1", "SAMEA114235870", "Universite Libre de Bruxelles", "ENA FIRST PUBLIC:2023 12 25T01:15:29Z|ENA LAST UPDATE:2023 12 25T01:15:29Z|External Id:SAMEA114235870|INSDC center name:Universite Libre de Bruxelles|INSDC first public:2023 12 25T01:15:29Z|INSDC last update:2023 12 25T01:15:29Z|INSDC status:public|Submitter Id:E MTAB 13223:Sample 1|age:5|broker name:ArrayExpress|cell type:leukocyte|collection date:not collected|common name:zebrafish|developmental stage:adult|genotype:wild type genotype|geographic location country and/or sea:not collected|individual:pool of 3 fish|isolate:not applicable|organism part:brain|sample name:E MTAB 13223:Sample 1|scientific name:Danio rerio|sex:male and female|strain:AB", null, null, null, null, null, null, null, null, "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "E MTAB 13223:Sample 1 p", "Sample 1 p", "A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "FACS sorting of cd45:Dsred cells. Brains from adult Tgp2ry12:GFP; cd45:DsRed fish n=3  were dissected in 0.9X Dulbecco\u2032s Phosphate Buffered Saline DPBS were triturated and treated with Liberase TM at 33\u00b0C for 30 45 minutes  fully dissociated using a syringe with a 26G needle and washed in 2% fetal bovine serum diluted in 0.9X DPBS. Cell suspensions were centrifuged at 290g 4\u00baC 10 min and filtered through a 40\u00b5m nylon mesh. Just before flow cytometry analysis  using calcein violet to exclude dead cells 1\u00b5M  Thermo Fisher. Flow cytometry acquisition and cell sorting was performed on a FACS ARIA II Becton Dickinson.  RNA Illumina", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "PAIRED", "ILLUMINA", "NextSeq 550", null, "ERP149944", "NextSeq 550 paired end sequencing; A single cell transcriptomic atlas reveals a new myeloid parenchymal population in the zebrafish brain", "ENA FIRST PUBLIC:2023 12 25|ENA LAST UPDATE:2023 12 25", "L41805_Track-81377_R1.fastq.gz L41805_Track-81377_R2.fastq.gz", "fastq fastq", 3184137096.0, 37906394.0, "E MTAB 13223:L41805 Track 81377", "0:28 1:56", "A:899943184;C:685571078;G:703849582;T:889228559;N:5544693", 28, 56, null, null, 899943184, 685571078, 703849582, 889228559, 5544693, "ERX11187331", "ERS16221143", "ERA26318932", "European Bioinformatics Institute|European Nucleotide Archive", "European Bioinformatics Institute|European Nucleotide Archive", 2, 0.00734, 0.90077, 0.00293, 0.20318, 0.99064, 0.82765, 0.47994, 0.53473, 28, 56, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-12-25", "Adult", "Adult", "Brain", "Nervous System"], [24967, "SRR25557924", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R2_001.fastq.gz", "fastq fastq fastq", 783833459.0, 6171917.0, "GSM7688796 r1", "0:8 1:28 2:91", "A:165863131;C:117191802;G:128146802;T:150173329;N:269383", 8, 28, 91, null, 165863131, 117191802, 128146802, 150173329, 269383, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89127, null, 0.221, null, 0.78317, null, 0.52195, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24968, "SRR25557925", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 7208342327.0, 56758601.0, "GSM7688796 r10", "0:8 1:28 2:91", "A:1518008610;C:1083228714;G:1190089577;T:1369772782;N:3933008", 8, 28, 91, null, 1518008610, 1083228714, 1190089577, 1369772782, 3933008, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88938, null, 0.21991, null, 0.79088, null, 0.53044, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24969, "SRR25557926", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 7337435414.0, 57775082.0, "GSM7688796 r11", "0:8 1:28 2:91", "A:1545051667;C:1103394058;G:1211577972;T:1395133564;N:2375201", 8, 28, 91, null, 1545051667, 1103394058, 1211577972, 1395133564, 2375201, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88966, null, 0.2211, null, 0.78961, null, 0.53156, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24970, "SRR25557927", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 7218240453.0, 56836539.0, "GSM7688796 r12", "0:8 1:28 2:91", "A:1521442237;C:1084242714;G:1191469584;T:1372277539;N:2692975", 8, 28, 91, null, 1521442237, 1084242714, 1191469584, 1372277539, 2692975, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8888, null, 0.2174, null, 0.79172, null, 0.53311, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24971, "SRR25557928", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R2_001.fastq.gz", "fastq fastq fastq", 774302109.0, 6096867.0, "GSM7688796 r2", "0:8 1:28 2:91", "A:163989105;C:115793536;G:126358958;T:148430473;N:242825", 8, 28, 91, null, 163989105, 115793536, 126358958, 148430473, 242825, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89371, null, 0.2215, null, 0.78253, null, 0.53282, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24972, "SRR25557929", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R2_001.fastq.gz", "fastq fastq fastq", 790430093.0, 6223859.0, "GSM7688796 r3", "0:8 1:28 2:91", "A:167465790;C:118269595;G:129024496;T:151447225;N:164063", 8, 28, 91, null, 167465790, 118269595, 129024496, 151447225, 164063, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89284, null, 0.21955, null, 0.78356, null, 0.53369, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24973, "SRR25557930", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5_S3_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R2_001.fastq.gz", "fastq fastq fastq", 778936974.0, 6133362.0, "GSM7688796 r4", "0:8 1:28 2:91", "A:165013558;C:116498474;G:127146290;T:149314806;N:162814", 8, 28, 91, null, 165013558, 116498474, 127146290, 149314806, 162814, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89195, null, 0.2209, null, 0.78196, null, 0.53121, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24974, "SRR25557931", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 4174244890.0, 32868070.0, "GSM7688796 r5", "0:8 1:28 2:91", "A:882060228;C:627098568;G:684890393;T:795503862;N:1441319", 8, 28, 91, null, 882060228, 627098568, 684890393, 795503862, 1441319, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89137, null, 0.22041, null, 0.77926, null, 0.52991, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24975, "SRR25557932", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 4150210902.0, 32678826.0, "GSM7688796 r6", "0:8 1:28 2:91", "A:878218072;C:623662333;G:679977758;T:790589138;N:1325865", 8, 28, 91, null, 878218072, 623662333, 679977758, 790589138, 1325865, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.89264, null, 0.22159, null, 0.78121, null, 0.52989, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24976, "SRR25557933", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 4198043547.0, 33055461.0, "GSM7688796 r7", "0:8 1:28 2:91", "A:888287475;C:630914324;G:688006958;T:799794077;N:1044117", 8, 28, 91, null, 888287475, 630914324, 688006958, 799794077, 1044117, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8912, null, 0.21889, null, 0.7822, null, 0.52774, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24977, "SRR25557934", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-2_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 4154784553.0, 32714839.0, "GSM7688796 r8", "0:8 1:28 2:91", "A:880520885;C:624179045;G:680076172;T:791297938;N:976309", 8, 28, 91, null, 880520885, 624179045, 680076172, 791297938, 976309, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8927, null, 0.22063, null, 0.78216, null, 0.5346, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24978, "SRR25557935", "SRX21286763", "SRS18536876", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 AllGFP S5", "GSM7688796", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 AllGFP S5", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688796", "GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq", "GSM7688796 r1", "GSM7688796", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_AllGFP-S5-3_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 7231973725.0, 56944675.0, "GSM7688796 r9", "0:8 1:28 2:91", "A:1522035629;C:1085783001;G:1195535350;T:1374885853;N:3725592", 8, 28, 91, null, 1522035629, 1085783001, 1195535350, 1374885853, 3725592, "SRX21286763", "SRS18536876", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.88833, null, 0.22076, null, 0.79056, null, 0.52744, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24979, "SRR25557936", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L001_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 699161670.0, 5505210.0, "GSM7688795 r1", "0:8 1:28 2:91", "A:149927051;C:100829317;G:116075103;T:133908276;N:234363", 8, 28, 91, null, 149927051, 100829317, 116075103, 133908276, 234363, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85081, null, 0.21257, null, 0.80426, null, 0.52779, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24980, "SRR25557937", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L002_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R2_001.fastq.gz", "fastq fastq fastq", 8174836330.0, 64368790.0, "GSM7688795 r10", "0:8 1:28 2:91", "A:1748532358;C:1187854561;G:1356279755;T:1560491610;N:4401606", 8, 28, 91, null, 1748532358, 1187854561, 1356279755, 1560491610, 4401606, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85069, null, 0.20989, null, 0.81049, null, 0.52069, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24981, "SRR25557938", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R2_001.fastq.gz", "fastq fastq fastq", 8307872640.0, 65416320.0, "GSM7688795 r11", "0:8 1:28 2:91", "A:1778112295;C:1207569588;G:1377139686;T:1587359299;N:2704252", 8, 28, 91, null, 1778112295, 1207569588, 1377139686, 1587359299, 2704252, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85151, null, 0.21054, null, 0.80813, null, 0.52675, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24982, "SRR25557939", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_I1_001.fastq.gz", "fastq fastq fastq", 8197523483.0, 64547429.0, "GSM7688795 r12", "0:8 1:28 2:91", "A:1755321680;C:1190415496;G:1359502433;T:1565500310;N:3076120", 8, 28, 91, null, 1755321680, 1190415496, 1359502433, 1565500310, 3076120, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.84956, null, 0.20968, null, 0.81113, null, 0.52382, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24983, "SRR25557940", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_I1_001.fastq.gz", "fastq fastq fastq", 690198899.0, 5434637.0, "GSM7688795 r2", "0:8 1:28 2:91", "A:147896108;C:99606794;G:114681063;T:132140792;N:227210", 8, 28, 91, null, 147896108, 99606794, 114681063, 132140792, 227210, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85228, null, 0.21261, null, 0.803, null, 0.52382, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24984, "SRR25557941", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R2_001.fastq.gz", "fastq fastq fastq", 702239515.0, 5529445.0, "GSM7688795 r3", "0:8 1:28 2:91", "A:150477574;C:101266550;G:116878466;T:134415299;N:141606", 8, 28, 91, null, 150477574, 101266550, 116878466, 134415299, 141606, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85364, null, 0.21344, null, 0.80346, null, 0.52158, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24985, "SRR25557942", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8_S2_L004_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R2_001.fastq.gz", "fastq fastq fastq", 694537346.0, 5468798.0, "GSM7688795 r4", "0:8 1:28 2:91", "A:148876263;C:100202441;G:115455212;T:132982029;N:144673", 8, 28, 91, null, 148876263, 100202441, 115455212, 132982029, 144673, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85238, null, 0.2138, null, 0.80379, null, 0.52691, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24986, "SRR25557943", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_I1_001.fastq.gz", "fastq fastq fastq", 4324997954.0, 34055102.0, "GSM7688795 r5", "0:8 1:28 2:91", "A:924471384;C:626141120;G:722856562;T:824050239;N:1494977", 8, 28, 91, null, 924471384, 626141120, 722856562, 824050239, 1494977, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85083, null, 0.21122, null, 0.80472, null, 0.524, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24987, "SRR25557944", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_I1_001.fastq.gz", "fastq fastq fastq", 4306232942.0, 33907346.0, "GSM7688795 r6", "0:8 1:28 2:91", "A:920615353;C:623090513;G:721201055;T:819291149;N:1370416", 8, 28, 91, null, 920615353, 623090513, 721201055, 819291149, 1370416, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85335, null, 0.21315, null, 0.80206, null, 0.51508, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24988, "SRR25557945", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L003_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_I1_001.fastq.gz", "fastq fastq fastq", 4348436312.0, 34239656.0, "GSM7688795 r7", "0:8 1:28 2:91", "A:930204208;C:629005094;G:727756813;T:827747478;N:1095103", 8, 28, 91, null, 930204208, 629005094, 727756813, 827747478, 1095103, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85369, null, 0.21216, null, 0.80503, null, 0.52219, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24989, "SRR25557946", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-2_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_I1_001.fastq.gz", "fastq fastq fastq", 4313915172.0, 33967836.0, "GSM7688795 r8", "0:8 1:28 2:91", "A:922742754;C:623127831;G:724345885;T:819819593;N:1037013", 8, 28, 91, null, 922742754, 623127831, 724345885, 819819593, 1037013, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.85456, null, 0.21236, null, 0.80223, null, 0.5216, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24990, "SRR25557947", "SRX21286762", "SRS18536875", "SRP453891", "PRJNA1003032", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq]", "GSE240239", "Transcriptome Analysis", "Background: V0v spinal interneurons are highly conserved  glutamatergic  commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However  we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks  we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings.  Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that  by this stage of development  evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons  or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons  plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly  we show that Hmx2/3a  repress dI2 interneuronal expression of skor1a and nefma  two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons  as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2  V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then  later  start to express markers of distinct types of inhibitory spinal interneurons  or motoneurons. Taken together  our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total  both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "MUTSU2 OtherGFP G8", "GSM7688795", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing", "MUTSU2 OtherGFP G8", "We performed demultiplexing and counts analysis as per the manufacturer\u2019s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or \u201cleaky\u201d cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA  graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting  using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type  evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow  we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.", "Spinal Cord", null, "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz\u2019s L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco\u2019s Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco\u2019s Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3\u2019 GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", "Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf  as confirmed by analysis of morphological criteria  including head trunk angle  head and eye size.", "tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1", "GSM7688795", "GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq", "GSM7688795 r1", "GSM7688795", "1", "Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf.  Embryos were deyolked  dissected and dissociated as described in GSE145916  with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord  and posteriorly  immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation  LK003150  trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 \u00b5l remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip  we passed each sample through a 40 \u00b5m Flowmi cell strainer Merck  BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation  samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific  21083027 + 0.5% FBS and stored on ice. Immediately before FACS  DAPI Merck  D9542 and Draq5 BioLegend  424101 were added at a final concentration of 5 \u00b5g/ml and 5 \u00b5M respectively.  FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al.  2008 with the following modifications. Ice cold samples were filtered through 35 \u00b5m mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon  352235. All FAC sorting and collection steps were performed at +4oC  using a 100 \u00b5m nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width  followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells.  EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made  pre chilled  90% methanol for HPLC  >99%  Merck  34860/10% Dulbecco's Phosphate Buffered Saline DPBS  No calcium  No magnesium  Merck  D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific  15250061 and a hemocytometer. Samples with intact  fully fixed cells  containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below.  To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells  we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next  we carefully removed the majority of the supernatant with a sterile p1000 tip  until approximately 100 \u00b5l remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made  pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline  no calcium  no magnesium Merck  D8537  1.0% UltraPure BSA ThermoFisher Scientific  AM2616  0.5 u/\u00b5l Roche Protector RNase Inhibitor Merck  3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step  we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 \u00b5l of supernatant. Using a sterile p200 tip  we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad  1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol \u201cMethanol Fixation of Cells for Single Cell RNA Sequencing\u201d https://www.10xgenomics.com  we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system  aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit  1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM  Library and Gel Bead Kit v3.1  10x Genomics  1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit  v2.5  150 cycles  20024907.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP453891", null, "loader:fastq load.py", "MUTSU2_OtherGFP-G8-3_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_I1_001.fastq.gz", "fastq fastq fastq", 8184961024.0, 64448512.0, "GSM7688795 r9", "0:8 1:28 2:91", "A:1748819280;C:1187802244;G:1360994184;T:1562975470;N:4223414", 8, 28, 91, null, 1748819280, 1187802244, 1360994184, 1562975470, 4223414, "SRX21286762", "SRS18536875", "SRA1688438", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.8493, null, 0.21072, null, 0.80967, null, 0.52593, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [28482, "SRR26266492", "SRX21975998", "SRS19051834", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  abca7 k/o   Ab42 injected", "GSM7819018", null, "source name:telencephalon|tissue:telencephalon|genotype:abca7 +/ |treatment:Ab42|geo loc name:missing|collection date:missing", "telencephalon  abca7 k/o   Ab42 injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:abca7 +/ |treatment:Ab42", "GSM7819018", "GSM7819018: telencephalon  abca7 k/o   Ab42 injected; Danio rerio; RNA Seq", "GSM7819018 r1", "GSM7819018", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP021_S12_L001_I1_001.fastq.gz CP021_S12_L001_I2_001.fastq.gz CP021_S12_L001_R1_001.fastq.gz CP021_S12_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 46637839920.0, 210080360.0, "GSM7819018 r1", "0:10 1:10 2:101 3:101", "A:12160340644;C:7050177030;G:7302497483;T:15922643298;N:574265", 10, 10, 101, 101, 12160340644, 7050177030, 7302497483, 15922643298, 574265, "SRX21975998", "SRS19051834", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00333, 0.89749, 0.00051, 0.26345, 0.99821, 0.73539, 0.54736, 0.5015, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28483, "SRR26266493", "SRX21975998", "SRS19051834", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  abca7 k/o   Ab42 injected", "GSM7819018", null, "source name:telencephalon|tissue:telencephalon|genotype:abca7 +/ |treatment:Ab42|geo loc name:missing|collection date:missing", "telencephalon  abca7 k/o   Ab42 injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:abca7 +/ |treatment:Ab42", "GSM7819018", "GSM7819018: telencephalon  abca7 k/o   Ab42 injected; Danio rerio; RNA Seq", "GSM7819018 r1", "GSM7819018", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP021_S12_L002_I1_001.fastq.gz CP021_S12_L002_I2_001.fastq.gz CP021_S12_L002_R1_001.fastq.gz CP021_S12_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 47673315186.0, 214744663.0, "GSM7819018 r2", "0:10 1:10 2:101 3:101", "A:12499321052;C:7231866994;G:7443419880;T:16203206561;N:607439", 10, 10, 101, 101, 12499321052, 7231866994, 7443419880, 16203206561, 607439, "SRX21975998", "SRS19051834", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00305, 0.89832, 0.00064, 0.26179, 0.99851, 0.73434, 0.53932, 0.50016, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28484, "SRR26266494", "SRX21975997", "SRS19051833", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  abca7 k/o   PBS injected", "GSM7819017", null, "source name:telencephalon|tissue:telencephalon|genotype:abca7 +/ |treatment:PBS|geo loc name:missing|collection date:missing", "telencephalon  abca7 k/o   PBS injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:abca7 +/ |treatment:PBS", "GSM7819017", "GSM7819017: telencephalon  abca7 k/o   PBS injected; Danio rerio; RNA Seq", "GSM7819017 r1", "GSM7819017", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP020_S11_L001_I1_001.fastq.gz CP020_S11_L001_I2_001.fastq.gz CP020_S11_L001_R1_001.fastq.gz CP020_S11_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 45135900918.0, 203314869.0, "GSM7819017 r1", "0:10 1:10 2:101 3:101", "A:11807801920;C:6876592307;G:7115059785;T:15269592915;N:556611", 10, 10, 101, 101, 11807801920, 6876592307, 7115059785, 15269592915, 556611, "SRX21975997", "SRS19051833", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00293, 0.90111, 0.00093, 0.28713, 0.99882, 0.73687, 0.58461, 0.50013, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28485, "SRR26266495", "SRX21975997", "SRS19051833", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  abca7 k/o   PBS injected", "GSM7819017", null, "source name:telencephalon|tissue:telencephalon|genotype:abca7 +/ |treatment:PBS|geo loc name:missing|collection date:missing", "telencephalon  abca7 k/o   PBS injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:abca7 +/ |treatment:PBS", "GSM7819017", "GSM7819017: telencephalon  abca7 k/o   PBS injected; Danio rerio; RNA Seq", "GSM7819017 r1", "GSM7819017", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP020_S11_L002_I1_001.fastq.gz CP020_S11_L002_I2_001.fastq.gz CP020_S11_L002_R1_001.fastq.gz CP020_S11_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 46689428280.0, 210312740.0, "GSM7819017 r2", "0:10 1:10 2:101 3:101", "A:12281435026;C:7136662243;G:7338982062;T:15725501987;N:592162", 10, 10, 101, 101, 12281435026, 7136662243, 7338982062, 15725501987, 592162, "SRX21975997", "SRS19051833", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00317, 0.90041, 0.00069, 0.28878, 0.99819, 0.73657, 0.60869, 0.49401, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28486, "SRR26266496", "SRX21975996", "SRS19051832", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  WT Ab42 injected", "GSM7819016", null, "source name:telencephalon|tissue:telencephalon|genotype:WT AB|treatment:Ab42|geo loc name:missing|collection date:missing", "telencephalon  WT Ab42 injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:WT AB|treatment:Ab42", "GSM7819016", "GSM7819016: telencephalon  WT Ab42 injected; Danio rerio; RNA Seq", "GSM7819016 r1", "GSM7819016", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP019_S10_L001_I1_001.fastq.gz CP019_S10_L001_I2_001.fastq.gz CP019_S10_L001_R1_001.fastq.gz CP019_S10_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 48132368784.0, 216812472.0, "GSM7819016 r1", "0:10 1:10 2:101 3:101", "A:12479944041;C:7377568938;G:7622029272;T:16315981376;N:595717", 10, 10, 101, 101, 12479944041, 7377568938, 7622029272, 16315981376, 595717, "SRX21975996", "SRS19051832", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00237, 0.90638, 0.00049, 0.23035, 0.99876, 0.73612, 0.50793, 0.50292, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28487, "SRR26266497", "SRX21975996", "SRS19051832", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  WT Ab42 injected", "GSM7819016", null, "source name:telencephalon|tissue:telencephalon|genotype:WT AB|treatment:Ab42|geo loc name:missing|collection date:missing", "telencephalon  WT Ab42 injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:WT AB|treatment:Ab42", "GSM7819016", "GSM7819016: telencephalon  WT Ab42 injected; Danio rerio; RNA Seq", "GSM7819016 r1", "GSM7819016", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP019_S10_L002_I1_001.fastq.gz CP019_S10_L002_I2_001.fastq.gz CP019_S10_L002_R1_001.fastq.gz CP019_S10_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 49675882836.0, 223765238.0, "GSM7819016 r2", "0:10 1:10 2:101 3:101", "A:12950211284;C:7640502677;G:7844712651;T:16764526831;N:624633", 10, 10, 101, 101, 12950211284, 7640502677, 7844712651, 16764526831, 624633, "SRX21975996", "SRS19051832", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.0034, 0.9058, 0.00062, 0.22634, 0.99825, 0.73423, 0.45714, 0.49934, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28488, "SRR26266498", "SRX21975995", "SRS19051831", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  WT  PBS injected", "GSM7819015", null, "source name:telencephalon|tissue:telencephalon|genotype:WT AB|treatment:PBS|geo loc name:missing|collection date:missing", "telencephalon  WT  PBS injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:WT AB|treatment:PBS", "GSM7819015", "GSM7819015: telencephalon  WT  PBS injected; Danio rerio; RNA Seq", "GSM7819015 r1", "GSM7819015", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP018_S9_L001_I1_001.fastq.gz CP018_S9_L001_I2_001.fastq.gz CP018_S9_L001_R1_001.fastq.gz CP018_S9_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 45948585420.0, 206975610.0, "GSM7819015 r1", "0:10 1:10 2:101 3:101", "A:12050390597;C:6986598890;G:7200535422;T:15570981408;N:566903", 10, 10, 101, 101, 12050390597, 6986598890, 7200535422, 15570981408, 566903, "SRX21975995", "SRS19051831", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00353, 0.89238, 0.00096, 0.30364, 0.99841, 0.73456, 0.59523, 0.49758, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28489, "SRR26266499", "SRX21975995", "SRS19051831", "SRP464334", "PRJNA1023540", "ABCA7 dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer's disease through BDNF/NGFR signaling", "GSE244550", "Transcriptome Analysis", "Genetic variants in ABCA7  an Alzheimer's disease AD associated gene  elevate AD risk  yet its functional relevance to the etiology is unclear. We generated a CRISPR Cas9 mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single cell transcriptomics in heterozygous abca7+/  knockout combined with A\u00df42 toxicity revealed that ABCA7 is crucial for neuropeptide Y NPY  brain derived neurotrophic factor BDNF  and nerve growth factor receptor NGFR expressions  which are crucial for synaptic integrity  astroglial proliferation  and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density  which are rescuable with ectopic NPY. In induced pluripotent stem cell derived human neurons exposed to A\u00df42  ABCA7 /  suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages  genetic variants in NPY associated with AD  and epigenetic changes in NPY  NGFR  and BDNF promoters linked to ABCA7 variants. Therefore  ABCA7 dependent NPY signaling via BDNF NGFR maintains synaptic integrity  implicating its impairment in increased AD risk through reduced brain resilience. Overall design: WT and abca7+/  zebrafish injected with Amyloid beta 42 peptide as described Bhatatrai et al. 2016 and cells were dissociated  sorted by FACS as described Cosacak et al. 2019. The single cell encapsulation and cDNA synthesis done by following 10X Genomics' workflows. The reads were aligned to zebrafish genome GRChZ 11  v 105 was used for gene annotation and assigning reads to genes. Cellranger software version 6.1.2 was used to process the data", null, "pubmed:39216475", null, "telencephalon  WT  PBS injected", "GSM7819015", null, "source name:telencephalon|tissue:telencephalon|genotype:WT AB|treatment:PBS|geo loc name:missing|collection date:missing", "telencephalon  WT  PBS injected", "The demultiplexing  barcoded processing  gene counting and aggregation were made using the 10x genomics' cellranger software version 6.1.2 https://support.10xgenomics.com/single cell gene expression/software/pipelines/latest/output/metrics The fastq files were aligned to zebrafish genome GRCz11 and ensemble transcripts from Ensembl Release 105 by using STAR. The BAM files were as input for Cell Ranger 10X genomics to generate processed data files that include gene names row names and cell names column names and counts. Further analysis done by using Seurat package in R. Assembly: GRCz11 Supplementary files format and content: filtered bc matrix outputs from Cell Ranger", "telencephalon", "WT AB and abca7 +/  knock out lines were injected with Amyloid beta 42 peptide  and PBS as control as descibed Bhatatrai et al. 2016.", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", "Zebrafish were kept in the re circulating system on a 14/10 h light/dark cycle  pH7.5  at 28\u00b0C \u00b11\u00b0C in groups of 20 animals per 2.8L.", "tissue:telencephalon|genotype:WT AB|treatment:PBS", "GSM7819015", "GSM7819015: telencephalon  WT  PBS injected; Danio rerio; RNA Seq", "GSM7819015 r1", "GSM7819015", "1", "Cells from zebrafish telencephalon were dissociated. Viability indicator dyes Sytox Blue Invitrogen  Cat No. S34857 and Dycle Ruby Invitrogen  Cat. No. V10309 were used to sort the cells by FACS. The library preparation was performed by 10X Genomics as per manufacture's protocol. 10X genomics", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP464334", null, "loader:fastq load.py", "CP018_S9_L002_I1_001.fastq.gz CP018_S9_L002_I2_001.fastq.gz CP018_S9_L002_R1_001.fastq.gz CP018_S9_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 47434063566.0, 213666953.0, "GSM7819015 r2", "0:10 1:10 2:101 3:101", "A:12506355665;C:7237420299;G:7414157832;T:16002190524;N:600186", 10, 10, 101, 101, 12506355665, 7237420299, 7414157832, 16002190524, 600186, "SRX21975995", "SRS19051831", "SRA1725563", "Neurology &amp; TAUB Institute, Columbia University", "Neurology & TAUB Institute, Columbia University", 2, 0.00327, 0.8932, 0.00092, 0.30198, 0.99831, 0.73401, 0.53488, 0.50008, 101, 101, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-10-03", "Undetermined", "Undetermined", "Brain", "Nervous System"], [28725, "SRR26623262", "SRX22323921", "SRS19374450", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  cirbpb scars", "GSM7875190", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  cirbpb scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875190", "GSM7875190: Lineage tracing rep1  cirbpb scars; Danio rerio; OTHER", "GSM7875190 r1", "GSM7875190", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_cirbpb_scar_R1.fastq.gz lin1_cirbpb_scar_R2.fastq.gz", "fastq fastq", 157576336.0, 949552.0, "GSM7875190 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323921", "SRS19374450", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00141, 0.78069, 0.00053, 0.00877, 0.99857, 0.97822, 0.33536, 0.05199, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28726, "SRR26623263", "SRX22323920", "SRS19374449", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  cfl1 scars", "GSM7875189", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  cfl1 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875189", "GSM7875189: Lineage tracing rep1  cfl1 scars; Danio rerio; OTHER", "GSM7875189 r1", "GSM7875189", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_cfl1_scar_R2.fastq.gz lin1_cfl1_scar_R1.fastq.gz", "fastq fastq", 381453696.0, 2184402.0, "GSM7875189 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323920", "SRS19374449", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0002, 0.91626, 0.00014, 0.00132, 0.99987, 0.99726, 0.16666, 0.56363, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28727, "SRR26623264", "SRX22323919", "SRS19374448", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  actb2 scars", "GSM7875188", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  actb2 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875188", "GSM7875188: Lineage tracing rep1  actb2 scars; Danio rerio; OTHER", "GSM7875188 r1", "GSM7875188", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_actb2_scar_R1.fastq.gz lin1_actb2_scar_R2.fastq.gz", "fastq fastq", 491682118.0, 2851156.0, "GSM7875188 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323919", "SRS19374448", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00051, 0.41901, 0.00032, 0.0029, 0.99945, 0.99182, 0.57575, 0.007, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28728, "SRR26623265", "SRX22323918", "SRS19374446", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  actb1 scars", "GSM7875187", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  actb1 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875187", "GSM7875187: Lineage tracing rep1  actb1 scars; Danio rerio; OTHER", "GSM7875187 r1", "GSM7875187", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_actb1_scar_R1.fastq.gz lin1_actb1_scar_R2.fastq.gz", "fastq fastq", 390405832.0, 2283319.0, "GSM7875187 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323918", "SRS19374446", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00038, 0.73548, 0.00017, 0.00057, 0.99963, 0.99571, 0.29729, 0.00243, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28729, "SRR26623266", "SRX22323917", "SRS19374447", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 23   telencephalon  Notch inhibition  scSLAMseq", "GSM7875186", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Notch inhibition DAPT|geo loc name:missing|collection date:missing", "Brain 23   telencephalon  Notch inhibition  scSLAMseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. Library strategy: scSLAM seq", "adult brain", "Notch inhibiton: zebrafish were incubated in a water bath containing system water with 50 \u00b5M DAPT Gamma Secretase Inhibitor  Sigma Aldrich for 48 hours.", "The samples were prepared according to a scSLAM seq protocol  adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts  200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected  and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 \u00b5l 100 mM IAA to 800 \u00b5l fixed sample was done overnight. The following day  the reaction was quanched with a quenching buffer containing 100 mM DTT  post which the sample was washed with a wash buffer and filtered though a 35 \u00b5m filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Notch inhibition DAPT", "GSM7875186", "GSM7875186: Brain 23   telencephalon  Notch inhibition  scSLAMseq; Danio rerio; OTHER", "GSM7875186 r1", "GSM7875186", "1", "The samples were prepared according to a scSLAM seq protocol  adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts  200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected  and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 \u00b5l 100 mM IAA to 800 \u00b5l fixed sample was done overnight. The following day  the reaction was quanched with a quenching buffer containing 100 mM DTT  post which the sample was washed with a wash buffer and filtered though a 35 \u00b5m filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "b23_ni_tre_R1.fastq.gz b23_ni_tre_R2.fastq.gz", "fastq fastq", 62392458570.0, 271271559.0, "GSM7875186 r1", "0:28 1:202", "A:18682905645;C:13783082970;G:14751373116;T:15161083446;N:14013393", 28, 202, null, null, 18682905645, 13783082970, 14751373116, 15161083446, 14013393, "SRX22323917", "SRS19374447", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01404, 0.82351, 0.00466, 0.1026, 0.99129, 0.86774, 0.36033, 0.67345, 28, 202, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28730, "SRR26623267", "SRX22323916", "SRS19374445", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 22   telencephalon  control  scSLAMseq", "GSM7875185", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Control DMSO|geo loc name:missing|collection date:missing", "Brain 22   telencephalon  control  scSLAMseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline. Library strategy: scSLAM seq", "adult brain", "Control for Notch inhibition: zebrafish were incubated in a water bath containing system water with 1:200 diluted DMSO for 48 hours.", "The samples were prepared according to a scSLAM seq protocol  adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts  200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected  and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 \u00b5l 100 mM IAA to 800 \u00b5l fixed sample was done overnight. The following day  the reaction was quanched with a quenching buffer containing 100 mM DTT  post which the sample was washed with a wash buffer and filtered though a 35 \u00b5m filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|treatment:Control DMSO", "GSM7875185", "GSM7875185: Brain 22   telencephalon  control  scSLAMseq; Danio rerio; OTHER", "GSM7875185 r1", "GSM7875185", "1", "The samples were prepared according to a scSLAM seq protocol  adapted from Neuschulz et al 2023. Briefly: in order to label nascent transcripts  200 mM 4sU was delivered to fish brains by intraventricular injection 6 hours prior to sample collection. The brains were then collected  and a single cell suspension was prepared by papain dissociation. The resulting cell suspension was fixed in 80% methanol and a conversion of 4sU using iodoactamide adding 111 \u00b5l 100 mM IAA to 800 \u00b5l fixed sample was done overnight. The following day  the reaction was quanched with a quenching buffer containing 100 mM DTT  post which the sample was washed with a wash buffer and filtered though a 35 \u00b5m filter. The sample was then loaded on a 10X Chromium Controller and processed according to the standard scRNA seq protocol. Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "b22_ni_con_R1.fastq.gz b22_ni_con_R2.fastq.gz", "fastq fastq", 59793114490.0, 259970063.0, "GSM7875185 r1", "0:28 1:202", "A:18600519530;C:12714987967;G:14147370465;T:14316863915;N:13372613", 28, 202, null, null, 18600519530, 12714987967, 14147370465, 14316863915, 13372613, "SRX22323916", "SRS19374445", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01402, 0.79448, 0.00498, 0.11405, 0.99074, 0.8518, 0.39874, 0.67082, 28, 202, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28731, "SRR26623268", "SRX22323915", "SRS19374444", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 20   sorted  scRNAseq", "GSM7875184", null, "source name:adult brain|tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]|geo loc name:missing|collection date:missing", "Brain 20   sorted  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]", "GSM7875184", "GSM7875184: Brain 20   sorted  scRNAseq; Danio rerio; RNA Seq", "GSM7875184 r1", "GSM7875184", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b20_gfap_R1.fastq.gz b20_gfap_R2.fastq.gz", "fastq fastq", 7798133320.0, 52690090.0, "GSM7875184 r1", "0:28 1:120", "A:2399592536;C:1516740530;G:1752554415;T:2127715674;N:1530165", 28, 120, null, null, 2399592536, 1516740530, 1752554415, 2127715674, 1530165, "SRX22323915", "SRS19374444", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0094, 0.86649, 0.00431, 0.28296, 0.9881, 0.77607, 0.40289, 0.50506, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28732, "SRR26623269", "SRX22323914", "SRS19374443", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 19   whole  scRNAseq", "GSM7875183", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 19   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875183", "GSM7875183: Brain 19   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875183 r1", "GSM7875183", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b19_tdmr_seqrun1_R1.fastq.gz b19_tdmr_seqrun1_R2.fastq.gz", "fastq fastq", 17234149570.0, 96821065.0, "GSM7875183 r1", "0:28 1:150", "A:5205716192;C:3547964017;G:3688127103;T:4785052541;N:7289717", 28, 150, null, null, 5205716192, 3547964017, 3688127103, 4785052541, 7289717, "SRX22323914", "SRS19374443", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0063, 0.8972, 0.00236, 0.22454, 0.99109, 0.79123, 0.52086, 0.54601, 28, 150, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28733, "SRR26623270", "SRX22323914", "SRS19374443", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 19   whole  scRNAseq", "GSM7875183", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 19   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875183", "GSM7875183: Brain 19   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875183 r1", "GSM7875183", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b19_tdmr_seqrun2_R1.fastq.gz b19_tdmr_seqrun2_R2.fastq.gz", "fastq fastq", 8080308788.0, 54596681.0, "GSM7875183 r2", "0:28 1:120", "A:2402417612;C:1698661627;G:1736701450;T:2240964763;N:1563336", 28, 120, null, null, 2402417612, 1698661627, 1736701450, 2240964763, 1563336, "SRX22323914", "SRS19374443", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00651, 0.88833, 0.00255, 0.2204, 0.99121, 0.7865, 0.532, 0.57621, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28734, "SRR26623271", "SRX22323913", "SRS19374442", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 18   rhombencephalon  scRNAseq", "GSM7875182", null, "source name:adult brain|tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 18   rhombencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]", "GSM7875182", "GSM7875182: Brain 18   rhombencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875182 r1", "GSM7875182", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b18_rhom_R1.fastq.gz b18_rhom_R2.fastq.gz", "fastq fastq", 18926303344.0, 127880428.0, "GSM7875182 r1", "0:28 1:120", "A:5713892455;C:3847687741;G:3928556839;T:5421348321;N:14817988", 28, 120, null, null, 5713892455, 3847687741, 3928556839, 5421348321, 14817988, "SRX22323913", "SRS19374442", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00496, 0.88436, 0.00243, 0.29801, 0.99285, 0.78433, 0.50833, 0.63318, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28735, "SRR26623272", "SRX22323912", "SRS19374441", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 18   mesencephalon  scRNAseq", "GSM7875181", null, "source name:adult brain|tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 18   mesencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]", "GSM7875181", "GSM7875181: Brain 18   mesencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875181 r1", "GSM7875181", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b18_mes_R1.fastq.gz b18_mes_R2.fastq.gz", "fastq fastq", 18106862272.0, 122343664.0, "GSM7875181 r1", "0:28 1:120", "A:5459512927;C:3636958859;G:3755613924;T:5240531488;N:14245074", 28, 120, null, null, 5459512927, 3636958859, 3755613924, 5240531488, 14245074, "SRX22323912", "SRS19374441", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00645, 0.88033, 0.00346, 0.34147, 0.99127, 0.76934, 0.48681, 0.60464, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28736, "SRR26623273", "SRX22323911", "SRS19374440", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 18   diencephalon  scRNAseq", "GSM7875180", null, "source name:adult brain|tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 18   diencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype  Tg[ubi:zebrabow M]", "GSM7875180", "GSM7875180: Brain 18   diencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875180 r1", "GSM7875180", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b18_dien_R1.fastq.gz b18_dien_R2.fastq.gz", "fastq fastq", 18653419612.0, 126036619.0, "GSM7875180 r1", "0:28 1:120", "A:5614496340;C:3825425595;G:3908771126;T:5290064867;N:14661684", 28, 120, null, null, 5614496340, 3825425595, 3908771126, 5290064867, 14661684, "SRX22323911", "SRS19374440", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00521, 0.89782, 0.00244, 0.25692, 0.99302, 0.77291, 0.47528, 0.62168, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28737, "SRR26623274", "SRX22323910", "SRS19374439", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 17   whole  scRNAseq", "GSM7875179", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 17   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875179", "GSM7875179: Brain 17   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875179 r1", "GSM7875179", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b17_tdmr_R1.fastq.gz b17_tdmr_R2.fastq.gz", "fastq fastq", 39604379236.0, 267597157.0, "GSM7875179 r1", "0:28 1:120", "A:12125317775;C:7792772449;G:8188608274;T:11493334367;N:4346371", 28, 120, null, null, 12125317775, 7792772449, 8188608274, 11493334367, 4346371, "SRX22323910", "SRS19374439", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00523, 0.88138, 0.0024, 0.31504, 0.99162, 0.75895, 0.40373, 0.49629, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28738, "SRR26623275", "SRX22323909", "SRS19374438", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 16   sorted  scRNAseq", "GSM7875178", null, "source name:adult brain|tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]|geo loc name:missing|collection date:missing", "Brain 16   sorted  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]", "GSM7875178", "GSM7875178: Brain 16   sorted  scRNAseq; Danio rerio; RNA Seq", "GSM7875178 r1", "GSM7875178", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b16_gfap_R2.fastq.gz b16_gfap_R1.fastq.gz", "fastq fastq", 5443014500.0, 36777125.0, "GSM7875178 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323909", "SRS19374438", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00627, 0.90735, 0.00227, 0.18428, 0.99115, 0.77242, 0.4456, 0.61483, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28739, "SRR26623276", "SRX22323908", "SRS19374437", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 15   whole  scRNAseq", "GSM7875177", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 15   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875177", "GSM7875177: Brain 15   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875177 r1", "GSM7875177", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b15_mult_R2.fastq.gz b15_mult_R1.fastq.gz", "fastq fastq", 11845962180.0, 80040285.0, "GSM7875177 r1", "0:28 1:120", "A:2976260508;C:2811030160;G:2912422943;T:3143400974;N:2847595", 28, 120, null, null, 2976260508, 2811030160, 2912422943, 3143400974, 2847595, "SRX22323908", "SRS19374437", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.08396, 0.93662, 0.02595, 0.16615, 0.93744, 0.71364, 0.29067, 0.56326, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28740, "SRR26623277", "SRX22323907", "SRS19374436", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 14   sorted  scRNAseq", "GSM7875176", null, "source name:adult brain|tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]|geo loc name:missing|collection date:missing", "Brain 14   sorted  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:sorted|cell type:mixed tissue dissociation|genotype:Tg[gfap:GFP]", "GSM7875176", "GSM7875176: Brain 14   sorted  scRNAseq; Danio rerio; RNA Seq", "GSM7875176 r1", "GSM7875176", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b14_gfap_R2.fastq.gz b14_gfap_R1.fastq.gz", "fastq fastq", 17138451800.0, 115800350.0, "GSM7875176 r1", "0:28 1:120", "A:4079957203;C:3974923538;G:4289276957;T:4791971757;N:2322345", 28, 120, null, null, 4079957203, 3974923538, 4289276957, 4791971757, 2322345, "SRX22323907", "SRS19374436", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.10093, 0.95317, 0.0314, 0.10551, 0.97104, 0.82487, 0.39909, 0.4491, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28741, "SRR26623278", "SRX22323906", "SRS19374435", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 13   whole  scRNAseq", "GSM7875175", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 13   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875175", "GSM7875175: Brain 13   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875175 r1", "GSM7875175", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b13_mult_R2.fastq.gz b13_mult_R1.fastq.gz", "fastq fastq", 14716484640.0, 94336440.0, "GSM7875175 r1", "0:28 1:128", "A:3656679229;C:3244551762;G:3483547888;T:4325660472;N:6045289", 28, 128, null, null, 3656679229, 3244551762, 3483547888, 4325660472, 6045289, "SRX22323906", "SRS19374435", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0334, 0.93779, 0.01344, 0.20291, 0.97654, 0.72991, 0.35896, 0.56645, 28, 128, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28742, "SRR26623279", "SRX22323905", "SRS19374434", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 12   whole  scRNAseq", "GSM7875174", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 12   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875174", "GSM7875174: Brain 12   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875174 r1", "GSM7875174", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b12_mult_R2.fastq.gz b12_mult_R1.fastq.gz", "fastq fastq", 12967118112.0, 83122552.0, "GSM7875174 r1", "0:28 1:128", "A:3152750696;C:2922178338;G:3166672580;T:3720207530;N:5308968", 28, 128, null, null, 3152750696, 2922178338, 3166672580, 3720207530, 5308968, "SRX22323905", "SRS19374434", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.06196, 0.94967, 0.02426, 0.19522, 0.97108, 0.73886, 0.33644, 0.62119, 28, 128, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28743, "SRR26623280", "SRX22323904", "SRS19374433", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 11   whole  scRNAseq", "GSM7875173", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 11   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875173", "GSM7875173: Brain 11   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875173 r1", "GSM7875173", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b11_mult_R1.fastq.gz b11_mult_R2.fastq.gz", "fastq fastq", 14974963824.0, 101182188.0, "GSM7875173 r1", "0:28 1:120", "A:3753936293;C:3687961330;G:3650849763;T:3879258880;N:2957558", 28, 120, null, null, 3753936293, 3687961330, 3650849763, 3879258880, 2957558, "SRX22323904", "SRS19374433", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.16926, 0.9633, 0.06452, 0.20524, 0.93545, 0.76274, 0.26732, 0.59389, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28744, "SRR26623281", "SRX22323903", "SRS19374431", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   telencephalon  scRNAseq", "GSM7875172", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875172", "GSM7875172: Brain 10   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875172 r1", "GSM7875172", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_tel_seqrun1_R2.fastq.gz b10_tel_seqrun1_R1.fastq.gz", "fastq fastq", 3731199140.0, 25210805.0, "GSM7875172 r1", "0:28 1:120", "A:1133663247;C:801786454;G:811411935;T:982912952;N:1424552", 28, 120, null, null, 1133663247, 801786454, 811411935, 982912952, 1424552, "SRX22323903", "SRS19374431", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01267, 0.92045, 0.00689, 0.20316, 0.99101, 0.80876, 0.4565, 0.44433, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28745, "SRR26623282", "SRX22323903", "SRS19374431", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   telencephalon  scRNAseq", "GSM7875172", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875172", "GSM7875172: Brain 10   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875172 r1", "GSM7875172", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_tel_seqrun2_R2.fastq.gz b10_tel_seqrun2_R1.fastq.gz", "fastq fastq", 506663940.0, 3423405.0, "GSM7875172 r2", null, null, null, null, null, null, null, null, null, null, null, "SRX22323903", "SRS19374431", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01234, 0.92119, 0.00661, 0.20424, 0.99176, 0.81156, 0.50821, 0.7283, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28746, "SRR26623283", "SRX22323902", "SRS19374432", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   mesencephalon  scRNAseq", "GSM7875171", null, "source name:adult brain|tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   mesencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875171", "GSM7875171: Brain 10   mesencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875171 r1", "GSM7875171", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_ot_seqrun1_R1.fastq.gz b10_ot_seqrun1_R2.fastq.gz", "fastq fastq", 13285400412.0, 89766219.0, "GSM7875171 r1", "0:28 1:120", "A:4046617952;C:2711660606;G:2795963083;T:3726083915;N:5074856", 28, 120, null, null, 4046617952, 2711660606, 2795963083, 3726083915, 5074856, "SRX22323902", "SRS19374432", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01072, 0.91211, 0.00629, 0.2341, 0.99111, 0.78985, 0.46996, 0.63777, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28747, "SRR26623284", "SRX22323902", "SRS19374432", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   mesencephalon  scRNAseq", "GSM7875171", null, "source name:adult brain|tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   mesencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:mesencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875171", "GSM7875171: Brain 10   mesencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875171 r1", "GSM7875171", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_ot_seqrun2_R1.fastq.gz b10_ot_seqrun2_R2.fastq.gz", "fastq fastq", 1804356800.0, 12191600.0, "GSM7875171 r2", "0:28 1:120", "A:547173169;C:367866083;G:382757407;T:506285738;N:274403", 28, 120, null, null, 547173169, 367866083, 382757407, 506285738, 274403, "SRX22323902", "SRS19374432", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0107, 0.91103, 0.00617, 0.23408, 0.99058, 0.79178, 0.4579, 0.63098, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28748, "SRR26623285", "SRX22323901", "SRS19374430", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   diencephalon  scRNAseq", "GSM7875170", null, "source name:adult brain|tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   diencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875170", "GSM7875170: Brain 10   diencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875170 r1", "GSM7875170", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_dien_seqrun1_R1.fastq.gz b10_dien_seqrun1_R2.fastq.gz", "fastq fastq", 16775453532.0, 113347659.0, "GSM7875170 r1", "0:28 1:120", "A:5122230488;C:3380526476;G:3500039522;T:4766209405;N:6447641", 28, 120, null, null, 5122230488, 3380526476, 3500039522, 4766209405, 6447641, "SRX22323901", "SRS19374430", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01031, 0.90264, 0.0062, 0.26938, 0.99107, 0.78283, 0.45373, 0.61666, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28749, "SRR26623286", "SRX22323901", "SRS19374430", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   diencephalon  scRNAseq", "GSM7875170", null, "source name:adult brain|tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   diencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875170", "GSM7875170: Brain 10   diencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875170 r1", "GSM7875170", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_dien_seqrun2_R1.fastq.gz b10_dien_seqrun2_R2.fastq.gz", "fastq fastq", 2278985844.0, 15398553.0, "GSM7875170 r2", "0:28 1:120", "A:693086062;C:458642995;G:479031763;T:647875888;N:349136", 28, 120, null, null, 693086062, 458642995, 479031763, 647875888, 349136, "SRX22323901", "SRS19374430", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01062, 0.90395, 0.0066, 0.27264, 0.99131, 0.78524, 0.45789, 0.61679, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28750, "SRR26623287", "SRX22323900", "SRS19374428", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   rhombencephalon  scRNAseq", "GSM7875169", null, "source name:adult brain|tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   rhombencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875169", "GSM7875169: Brain 10   rhombencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875169 r1", "GSM7875169", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_cer_seqrun1_R1.fastq.gz b10_cer_seqrun1_R2.fastq.gz", "fastq fastq", 16240869244.0, 109735603.0, "GSM7875169 r1", "0:28 1:120", "A:4994572796;C:3251585988;G:3395902822;T:4592558542;N:6249096", 28, 120, null, null, 4994572796, 3251585988, 3395902822, 4592558542, 6249096, "SRX22323900", "SRS19374428", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0128, 0.89159, 0.00813, 0.3597, 0.98991, 0.82278, 0.44977, 0.60211, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28751, "SRR26623288", "SRX22323900", "SRS19374428", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 10   rhombencephalon  scRNAseq", "GSM7875169", null, "source name:adult brain|tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype|geo loc name:missing|collection date:missing", "Brain 10   rhombencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:wildtype", "GSM7875169", "GSM7875169: Brain 10   rhombencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875169 r1", "GSM7875169", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b10_cer_seqrun2_R1.fastq.gz b10_cer_seqrun2_R2.fastq.gz", "fastq fastq", 2208762212.0, 14924069.0, "GSM7875169 r2", "0:28 1:120", "A:677215802;C:441481145;G:464737322;T:624989898;N:338045", 28, 120, null, null, 677215802, 441481145, 464737322, 624989898, 338045, "SRX22323900", "SRS19374428", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0127, 0.89091, 0.00771, 0.35998, 0.9893, 0.82432, 0.41448, 0.59902, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28752, "SRR26623289", "SRX22323899", "SRS19374429", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 9   telencephalon  scRNAseq", "GSM7875168", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 9   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875168", "GSM7875168: Brain 9   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875168 r1", "GSM7875168", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP469552", null, "loader:fastq load.py", "b9_tel_R1.fastq.gz b9_tel_R2.fastq.gz", "fastq fastq", 15193441000.0, 122527750.0, "GSM7875168 r1", "0:26 1:98", "A:4477414579;C:3090884741;G:3307200604;T:4310709839;N:7231237", 26, 98, null, null, 4477414579, 3090884741, 3307200604, 4310709839, 7231237, "SRX22323899", "SRS19374429", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00277, 0.88718, 0.00117, 0.25579, 0.99586, 0.79657, 0.30868, 0.52408, 26, 98, "T", "B", "sc-like readlen", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28753, "SRR26623290", "SRX22323898", "SRS19374427", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 8   whole  scRNAseq", "GSM7875167", null, "source name:adult brain|tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 8   whole  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:whole|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875167", "GSM7875167: Brain 8   whole  scRNAseq; Danio rerio; RNA Seq", "GSM7875167 r1", "GSM7875167", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b8_mult_R1.fastq.gz b8_mult_R2.fastq.gz", "fastq fastq", 16275377550.0, 108502517.0, "GSM7875167 r1", "0:26 1:124", "A:5027003801;C:3172670099;G:3435240300;T:4638692244;N:1771106", 26, 124, null, null, 5027003801, 3172670099, 3435240300, 4638692244, 1771106, "SRX22323898", "SRS19374427", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.02156, 0.88716, 0.00827, 0.36741, 0.97224, 0.83228, 0.41427, 0.52965, 26, 124, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28754, "SRR26623291", "SRX22323897", "SRS19374426", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep2  ube2e1 scars", "GSM7875196", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep2  ube2e1 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875196", "GSM7875196: Lineage tracing rep2  ube2e1 scars; Danio rerio; OTHER", "GSM7875196 r1", "GSM7875196", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "lin2_ube2e1_scar_R1.fastq.gz lin2_ube2e1_scar_R2.fastq.gz", "fastq fastq", 213092161.0, 1190459.0, "GSM7875196 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323897", "SRS19374426", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00752, 0.90776, 0.00286, 0.01739, 0.99346, 0.95444, 0.46002, 0.96817, 28, 151, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28755, "SRR26623292", "SRX22323896", "SRS19374425", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep2  rpl39 scars", "GSM7875195", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep2  rpl39 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875195", "GSM7875195: Lineage tracing rep2  rpl39 scars; Danio rerio; OTHER", "GSM7875195 r1", "GSM7875195", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "lin2_rpl39_scar_R2.fastq.gz lin2_rpl39_scar_R1.fastq.gz", "fastq fastq", 1023403502.0, 5717338.0, "GSM7875195 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323896", "SRS19374425", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00086, 0.83744, 0.0003, 0.00337, 0.99845, 0.97057, 0.26605, 0.40241, 28, 151, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28756, "SRR26623293", "SRX22323895", "SRS19374424", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep2  cirbpb scars", "GSM7875194", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep2  cirbpb scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875194", "GSM7875194: Lineage tracing rep2  cirbpb scars; Danio rerio; OTHER", "GSM7875194 r1", "GSM7875194", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "lin2_cirbpb_scar_R1.fastq.gz lin2_cirbpb_scar_R2.fastq.gz", "fastq fastq", 276120388.0, 1542572.0, "GSM7875194 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323895", "SRS19374424", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00206, 0.80328, 0.00078, 0.00662, 0.9975, 0.98039, 0.55421, 0.0258, 28, 151, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28757, "SRR26623294", "SRX22323894", "SRS19374423", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep2  cfl1 scars", "GSM7875193", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep2  cfl1 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875193", "GSM7875193: Lineage tracing rep2  cfl1 scars; Danio rerio; OTHER", "GSM7875193 r1", "GSM7875193", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP469552", null, "loader:fastq load.py", "lin2_cfl1_scar_R1.fastq.gz lin2_cfl1_scar_R2.fastq.gz", "fastq fastq", 791318009.0, 4420771.0, "GSM7875193 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323894", "SRS19374423", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00067, 0.46079, 0.00055, 0.16353, 0.99965, 0.99332, 0.52631, 0.44939, 28, 151, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28758, "SRR26623295", "SRX22323893", "SRS19374421", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  ube2e1 scars", "GSM7875192", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  ube2e1 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875192", "GSM7875192: Lineage tracing rep1  ube2e1 scars; Danio rerio; OTHER", "GSM7875192 r1", "GSM7875192", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_ube2e1_scar_R1.fastq.gz lin1_ube2e1_scar_R2.fastq.gz", "fastq fastq", 99407618.0, 601781.0, "GSM7875192 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323893", "SRS19374421", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00657, 0.84532, 0.00205, 0.03049, 0.99537, 0.95286, 0.43306, 0.19203, 28, 150, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28759, "SRR26623296", "SRX22323892", "SRS19374422", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Lineage tracing rep1  rpl39 scars", "GSM7875191", null, "source name:adult brain|tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Lineage tracing rep1  rpl39 scars", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875191", "GSM7875191: Lineage tracing rep1  rpl39 scars; Danio rerio; OTHER", "GSM7875191 r1", "GSM7875191", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "lin1_rpl39_scar_R1.fastq.gz lin1_rpl39_scar_R2.fastq.gz", "fastq fastq", 401898718.0, 2276836.0, "GSM7875191 r1", null, null, null, null, null, null, null, null, null, null, null, "SRX22323892", "SRS19374422", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00037, 0.2642, 0.00015, 0.00086, 0.99922, 0.99026, 0.23076, 0.42455, 28, 120, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28760, "SRR26623297", "SRX22323891", "SRS19374420", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 7   telencephalon  scRNAseq", "GSM7875166", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 7   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875166", "GSM7875166: Brain 7   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875166 r1", "GSM7875166", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP469552", null, "loader:fastq load.py", "b7_tel_R1.fastq.gz b7_tel_R2.fastq.gz", "fastq fastq", 7764380900.0, 62615975.0, "GSM7875166 r1", "0:26 1:98", "A:2261256925;C:1693767854;G:1688226491;T:2120472828;N:656802", 26, 98, null, null, 2261256925, 1693767854, 1688226491, 2120472828, 656802, "SRX22323891", "SRS19374420", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.01116, 0.89516, 0.0091, 0.16599, 0.99356, 0.80499, 0.47969, 0.71564, 26, 98, "T", "B", "sc-like readlen", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28761, "SRR26623298", "SRX22323890", "SRS19374418", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 7   diencephalon  scRNAseq", "GSM7875165", null, "source name:adult brain|tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 7   diencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:diencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875165", "GSM7875165: Brain 7   diencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875165 r1", "GSM7875165", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP469552", null, "loader:fastq load.py", "b7_dien_R1.fastq.gz b7_dien_R2.fastq.gz", "fastq fastq", 7513293052.0, 60591073.0, "GSM7875165 r1", "0:26 1:98", "A:2197597087;C:1625633976;G:1631364985;T:2058064795;N:632209", 26, 98, null, null, 2197597087, 1625633976, 1631364985, 2058064795, 632209, "SRX22323890", "SRS19374418", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00898, 0.89596, 0.00758, 0.17001, 0.99588, 0.81235, 0.32967, 0.59095, 26, 98, "T", "B", "sc-like readlen", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28762, "SRR26623299", "SRX22323889", "SRS19374417", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 7   rhombencephalon  scRNAseq", "GSM7875164", null, "source name:adult brain|tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 7   rhombencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:rhombencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875164", "GSM7875164: Brain 7   rhombencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875164 r1", "GSM7875164", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP469552", null, "loader:fastq load.py", "b7_cer_R1.fastq.gz b7_cer_R2.fastq.gz", "fastq fastq", 4549622496.0, 36690504.0, "GSM7875164 r1", "0:26 1:98", "A:1326792866;C:971674770;G:1022429173;T:1227318552;N:1407135", 26, 98, null, null, 1326792866, 971674770, 1022429173, 1227318552, 1407135, "SRX22323889", "SRS19374417", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.02024, 0.89648, 0.01794, 0.19303, 0.99283, 0.81542, 0.39821, 0.6673, 26, 98, "T", "B", "sc-like readlen", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28763, "SRR26623300", "SRX22323888", "SRS19374416", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 6   telencephalon  scRNAseq", "GSM7875163", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 6   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875163", "GSM7875163: Brain 6   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875163 r1", "GSM7875163", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP469552", null, "loader:fastq load.py", "b6_tel_R2.fastq.gz b6_tel_R1.fastq.gz", "fastq fastq", 35200219264.0, 283872736.0, "GSM7875163 r1", "0:26 1:98", "A:10338159874;C:7412176847;G:7655217994;T:9778922063;N:15742486", 26, 98, null, null, 10338159874, 7412176847, 7655217994, 9778922063, 15742486, "SRX22323888", "SRS19374416", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00528, 0.8891, 0.00408, 0.20496, 0.99577, 0.78567, 0.41949, 0.62904, 26, 98, "T", "B", "sc-like readlen", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28764, "SRR26623301", "SRX22323887", "SRS19374415", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 5   telencephalon  scRNAseq", "GSM7875162", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 5   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875162", "GSM7875162: Brain 5   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875162 r1", "GSM7875162", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b5_tel_R1.fastq.gz b5_tel_R2.fastq.gz", "fastq fastq", 15272326692.0, 110669034.0, "GSM7875162 r1", "0:28 1:110", "A:4657813514;C:3027716754;G:3480263590;T:4105516942;N:1015892", 28, 110, null, null, 4657813514, 3027716754, 3480263590, 4105516942, 1015892, "SRX22323887", "SRS19374415", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00158, 0.89454, 0.00083, 0.25984, 0.99752, 0.80413, 0.57553, 0.62731, 28, 110, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28765, "SRR26623302", "SRX22323886", "SRS19374419", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 4   telencephalon  scRNAseq", "GSM7875161", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 4   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875161", "GSM7875161: Brain 4   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875161 r1", "GSM7875161", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina MiniSeq", null, "SRP469552", null, "loader:fastq load.py", "b4_tel_R1.fastq.gz b4_tel_R2.fastq.gz", "fastq fastq", 4538898000.0, 36023000.0, "GSM7875161 r1", "0:26 1:100", "A:1403578459;C:926366830;G:979609921;T:1228675807;N:666983", 26, 100, null, null, 1403578459, 926366830, 979609921, 1228675807, 666983, "SRX22323886", "SRS19374419", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.0031, 0.90947, 0.0017, 0.32332, 0.99592, 0.82881, 0.4664, 0.6686, 26, 100, "T", "B", "sc-like readlen", "illumina", "miseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28766, "SRR26623303", "SRX22323885", "SRS19374414", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 3   telencephalon  scRNAseq", "GSM7875160", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 3   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875160", "GSM7875160: Brain 3   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875160 r1", "GSM7875160", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b3_tel_R2.fastq.gz b3_tel_R1.fastq.gz", "fastq fastq", 14555700180.0, 173282145.0, "GSM7875160 r1", "0:26 1:58", "A:4218664834;C:3011617868;G:3387306043;T:3936116224;N:1995211", 26, 58, null, null, 4218664834, 3011617868, 3387306043, 3936116224, 1995211, "SRX22323885", "SRS19374414", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.00416, 0.90236, 0.00175, 0.29743, 0.99247, 0.79539, 0.34888, 0.56327, 26, 58, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28767, "SRR26623304", "SRX22323884", "SRS19374412", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 2   telencephalon  scRNAseq", "GSM7875159", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 2   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875159", "GSM7875159: Brain 2   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875159 r1", "GSM7875159", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b2_tel_I1.fastq.gz b2_tel_R1.fastq.gz b2_tel_R2.fastq.gz", "fastq fastq fastq", 18703678137.0, 144989753.0, "GSM7875159 r1", "0:14 1:95 2:20", "A:4322399040;C:2535898192;G:2691972122;T:4223349713;N:407468", 14, 95, 20, null, 4322399040, 2535898192, 2691972122, 4223349713, 407468, "SRX22323884", "SRS19374412", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 1, 0.91473, null, 0.25102, null, 0.79231, null, 0.54865, null, 95, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [28768, "SRR26623305", "SRX22323883", "SRS19374413", "SRP469552", "PRJNA1034159", "Dissecting the spatiotemporal diversity of adult neural stem cells", "GSE246714", "Other", "Adult stem cells are important for tissue turnover and regeneration. However  in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here  we dissected the diversity of neural stem cells in the adult zebrafish brain  an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells  with some subtypes being restricted to a single brain region  while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation  with different states being involved in proliferative and non proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes. Overall design: Single cell RNA seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain  FACS sorted cells GFP positive cells from gfap:GFP line or a dissected single brain region  as indicated in the sample title. CRISPR based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.", null, "pubmed:38365956", null, "Brain 1   telencephalon  scRNAseq", "GSM7875158", null, "source name:adult brain|tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]|geo loc name:missing|collection date:missing", "Brain 1   telencephalon  scRNAseq", "The transcriptome libraries were demultiplexed and mapped with CellRanger 6.1.1. In order to reduce batch effect  the gene expression matrices were processed with the SoupX tool Young & Behjati  2020 for removing ambient RNA contamination. A subset of libraries was also mapped with Velocyto v0.17.17 La Manno et al  2018 to create count matrices for unspliced and spliced molecules. The targeted lineage tracing libraries were aligned using bwa mem3 v.0.7.12 to individual references of endogenous genes used in the lineage tracing experiment actb1  actb2  cfl1  cirbpb  rpl39 and ube2e1. The scars were filtered with a custom pipeline to eliminate sequences that originate from PCR or sequencing errors. For custom code and further downstream analysis  see: https://github.com/nimitic/radial glia. Assembly: GRCz11 Supplementary files format and content: Tab separated barcode files and matrix files along with the tab separated gene file supplementary file are the output of CellRanger mapping  further processed by the SoupX package for ambient RNA removal. Supplementary files format and content: Loom files are the output of velocyto mapping distinguishing spliced and unspliced mRNAs. Supplementary files format and content: filtered scars.csv files contain the output of a custom scar filtering pipeline.", "adult brain", null, "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "tissue:adult brain|tissue region:telencephalon|cell type:mixed tissue dissociation|genotype:Tg[ubi:zebrabow M]", "GSM7875158", "GSM7875158: Brain 1   telencephalon  scRNAseq; Danio rerio; RNA Seq", "GSM7875158 r1", "GSM7875158", "1", "Adult zebrafish brain tissue was extracted  optionally dissected to select a specific region of interest telencephalon  diencephalon  mesencephalon or rhombencephalon and then dissociated with a trypsin protocol. Briefly  the samples were incubated in 750 \u00b5l of HBSS glucose solution and dissocated with 15 \u00b5l of 0.05% trypsin EDTA Gibco for 30 minutes at 37\u00b0C with intermittent mixing. The disociation was stopped by addition of 750 \u00b5l of a BSA EBSS HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4\u00b0C. The sample was filtered through a 100 \u00b5m filter  washed with cold HBSS and resuspended in 100 200 \u00b5l HBSS with 0.05% BSA  then filtered through a 35 \u00b5m filter and loaded on the 10x Chromium Controller. For the scSLAM seq samples  a papain dissociation protocol Worthington kit  according to manufecturer's instructions was used followed by an adapted scSLAM seq protocol for labeling nascent transcripts see sample specific entry. The standard transcriptome libraries were constructed according to the manufacturer's protocol 10X Genomics. For targeted libraries of scars used for lineage tracing  the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target  specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit  so they could be sequenced on the same platform.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP469552", null, "loader:fastq load.py", "b1_tel_R2.fastq.gz b1_tel_R1.fastq.gz b1_tel_I1.fastq.gz", "fastq fastq fastq", 21073338957.0, 132536723.0, "GSM7875158 r1", "0:14 1:95 2:50", "A:4785729675;C:2770008572;G:5684239548;T:5977128662;N:718378", 14, 95, 50, null, 4785729675, 2770008572, 5684239548, 5977128662, 718378, "SRX22323883", "SRS19374413", "SRA1743007", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", "Junker Lab, Berlin Institute for Medical Systems Biology, Max-Delbr\u00fcck-Center for Molecular Medicine", 2, 0.90262, 0.10312, 0.25979, 0.09812, 0.81182, 0.99951, 0.5478, 0.24137, 95, 50, "B", "T", "mate2 technical by mapping diff", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Germany", "2023-10-31", "Adult", "Adult", "Brain", "Nervous System"], [30256, "SRR27747506", "SRX23412819", "SRS20268140", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Late born vestibular neurons  replicate 2", "GSM8038041", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede|geo loc name:missing|collection date:missing", "Late born vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede", "GSM8038041", "GSM8038041: Late born vestibular neurons  replicate 2; Danio rerio; RNA Seq", "GSM8038041 r1", "GSM8038041", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Green_S19_L001_I1_001.fastq.gz Green_S19_L001_I2_001.fastq.gz Green_S19_L001_R1_001.fastq.gz Green_S19_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 4923159660.0, 35675070.0, "GSM8038041 r1", "0:10 1:10 2:28 3:90", "A:1637293866;C:391317121;G:716609880;T:465465343;N:70090", 10, 10, 28, 90, 1637293866, 391317121, 716609880, 465465343, 70090, "SRX23412819", "SRS20268140", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30257, "SRR27747507", "SRX23412819", "SRS20268140", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Late born vestibular neurons  replicate 2", "GSM8038041", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede|geo loc name:missing|collection date:missing", "Late born vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede", "GSM8038041", "GSM8038041: Late born vestibular neurons  replicate 2; Danio rerio; RNA Seq", "GSM8038041 r1", "GSM8038041", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Green_S19_L002_I1_001.fastq.gz Green_S19_L002_I2_001.fastq.gz Green_S19_L002_R1_001.fastq.gz Green_S19_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 4858633206.0, 35207487.0, "GSM8038041 r2", "0:10 1:10 2:28 3:90", "A:1614338875;C:386568724;G:706765488;T:460927318;N:73425", 10, 10, 28, 90, 1614338875, 386568724, 706765488, 460927318, 73425, "SRX23412819", "SRS20268140", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30258, "SRR27747508", "SRX23412819", "SRS20268140", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Late born vestibular neurons  replicate 2", "GSM8038041", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede|geo loc name:missing|collection date:missing", "Late born vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede", "GSM8038041", "GSM8038041: Late born vestibular neurons  replicate 2; Danio rerio; RNA Seq", "GSM8038041 r1", "GSM8038041", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Green_S19_L003_I1_001.fastq.gz Green_S19_L003_I2_001.fastq.gz Green_S19_L003_R1_001.fastq.gz Green_S19_L003_R2_001.fastq.gz", "fastq fastq fastq fastq", 4458723696.0, 32309592.0, "GSM8038041 r3", "0:10 1:10 2:28 3:90", "A:1463412697;C:359363166;G:655814899;T:429192204;N:80314", 10, 10, 28, 90, 1463412697, 359363166, 655814899, 429192204, 80314, "SRX23412819", "SRS20268140", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30259, "SRR27747509", "SRX23412819", "SRS20268140", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Late born vestibular neurons  replicate 2", "GSM8038041", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede|geo loc name:missing|collection date:missing", "Late born vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born post 36 hpf green unconverted Kaede", "GSM8038041", "GSM8038041: Late born vestibular neurons  replicate 2; Danio rerio; RNA Seq", "GSM8038041 r1", "GSM8038041", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Green_S19_L004_I1_001.fastq.gz Green_S19_L004_I2_001.fastq.gz Green_S19_L004_R1_001.fastq.gz Green_S19_L004_R2_001.fastq.gz", "fastq fastq fastq fastq", 4425208602.0, 32066729.0, "GSM8038041 r4", "0:10 1:10 2:28 3:90", "A:1452148554;C:355763806;G:651302168;T:426718847;N:72235", 10, 10, 28, 90, 1452148554, 355763806, 651302168, 426718847, 72235, "SRX23412819", "SRS20268140", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30260, "SRR27747510", "SRX23412818", "SRS20268139", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Early born vestibular neurons  replicate 1", "GSM8038040", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede|geo loc name:missing|collection date:missing", "Early born vestibular neurons  replicate 1", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede", "GSM8038040", "GSM8038040: Early born vestibular neurons  replicate 1; Danio rerio; RNA Seq", "GSM8038040 r1", "GSM8038040", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Red_S20_L001_I1_001.fastq.gz Red_S20_L001_I2_001.fastq.gz Red_S20_L001_R1_001.fastq.gz Red_S20_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 5032560816.0, 36467832.0, "GSM8038040 r1", "0:10 1:10 2:28 3:90", "A:1626033872;C:414606578;G:755475703;T:485911557;N:77170", 10, 10, 28, 90, 1626033872, 414606578, 755475703, 485911557, 77170, "SRX23412818", "SRS20268139", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30261, "SRR27747511", "SRX23412818", "SRS20268139", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Early born vestibular neurons  replicate 1", "GSM8038040", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede|geo loc name:missing|collection date:missing", "Early born vestibular neurons  replicate 1", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede", "GSM8038040", "GSM8038040: Early born vestibular neurons  replicate 1; Danio rerio; RNA Seq", "GSM8038040 r1", "GSM8038040", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Red_S20_L002_I1_001.fastq.gz Red_S20_L002_I2_001.fastq.gz Red_S20_L002_R1_001.fastq.gz Red_S20_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 4998616680.0, 36221860.0, "GSM8038040 r2", "0:10 1:10 2:28 3:90", "A:1613203224;C:412246760;G:750087642;T:484348061;N:81713", 10, 10, 28, 90, 1613203224, 412246760, 750087642, 484348061, 81713, "SRX23412818", "SRS20268139", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30262, "SRR27747512", "SRX23412818", "SRS20268139", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Early born vestibular neurons  replicate 1", "GSM8038040", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede|geo loc name:missing|collection date:missing", "Early born vestibular neurons  replicate 1", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede", "GSM8038040", "GSM8038040: Early born vestibular neurons  replicate 1; Danio rerio; RNA Seq", "GSM8038040 r1", "GSM8038040", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Red_S20_L003_I1_001.fastq.gz Red_S20_L003_I2_001.fastq.gz Red_S20_L003_R1_001.fastq.gz Red_S20_L003_R2_001.fastq.gz", "fastq fastq fastq fastq", 4628879490.0, 33542605.0, "GSM8038040 r3", "0:10 1:10 2:28 3:90", "A:1475662550;C:386397974;G:701772418;T:454914612;N:86896", 10, 10, 28, 90, 1475662550, 386397974, 701772418, 454914612, 86896, "SRX23412818", "SRS20268139", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30263, "SRR27747513", "SRX23412818", "SRS20268139", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "Early born vestibular neurons  replicate 1", "GSM8038040", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede|geo loc name:missing|collection date:missing", "Early born vestibular neurons  replicate 1", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:Neurons born before 36 hpf red photoconverted Kaede", "GSM8038040", "GSM8038040: Early born vestibular neurons  replicate 1; Danio rerio; RNA Seq", "GSM8038040 r1", "GSM8038040", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Red_S20_L004_I1_001.fastq.gz Red_S20_L004_I2_001.fastq.gz Red_S20_L004_R1_001.fastq.gz Red_S20_L004_R2_001.fastq.gz", "fastq fastq fastq fastq", 4582808190.0, 33208755.0, "GSM8038040 r4", "0:10 1:10 2:28 3:90", "A:1460627048;C:381772307;G:695044521;T:451264941;N:79133", 10, 10, 28, 90, 1460627048, 381772307, 695044521, 451264941, 79133, "SRX23412818", "SRS20268139", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30264, "SRR27747514", "SRX23412817", "SRS20268138", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 3", "GSM8038039", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 3", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a", "GSM8038039", "GSM8038039: All vestibular neurons  replicate 3; Danio rerio; RNA Seq", "GSM8038039 r1", "GSM8038039", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Zebrafish-Replicate3_S3_L001_I1_001.fastq.gz Zebrafish-Replicate3_S3_L001_I2_001.fastq.gz Zebrafish-Replicate3_S3_L001_R1_001.fastq.gz Zebrafish-Replicate3_S3_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 10560652880.0, 75975920.0, "GSM8038039 r1", "0:10 1:10 2:28 3:91", "A:2623816838;C:1175510072;G:1638990770;T:1474946754;N:544286", 10, 10, 28, 91, 2623816838, 1175510072, 1638990770, 1474946754, 544286, "SRX23412817", "SRS20268138", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30265, "SRR27747515", "SRX23412817", "SRS20268138", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 3", "GSM8038039", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 3", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a", "GSM8038039", "GSM8038039: All vestibular neurons  replicate 3; Danio rerio; RNA Seq", "GSM8038039 r1", "GSM8038039", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Zebrafish-Replicate3_S3_L002_I1_001.fastq.gz Zebrafish-Replicate3_S3_L002_I2_001.fastq.gz Zebrafish-Replicate3_S3_L002_R1_001.fastq.gz Zebrafish-Replicate3_S3_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 10571317933.0, 76052647.0, "GSM8038039 r2", "0:10 1:10 2:28 3:91", "A:2631345994;C:1175630391;G:1641110007;T:1472183634;N:520851", 10, 10, 28, 91, 2631345994, 1175630391, 1641110007, 1472183634, 520851, "SRX23412817", "SRS20268138", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30266, "SRR27747516", "SRX23412817", "SRS20268138", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 3", "GSM8038039", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 3", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a", "GSM8038039", "GSM8038039: All vestibular neurons  replicate 3; Danio rerio; RNA Seq", "GSM8038039 r1", "GSM8038039", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Zebrafish-Replicate3_S3_L003_I1_001.fastq.gz Zebrafish-Replicate3_S3_L003_I2_001.fastq.gz Zebrafish-Replicate3_S3_L003_R1_001.fastq.gz Zebrafish-Replicate3_S3_L003_R2_001.fastq.gz", "fastq fastq fastq fastq", 10457106081.0, 75230979.0, "GSM8038039 r3", "0:10 1:10 2:28 3:91", "A:2588687752;C:1168371662;G:1623033341;T:1465362298;N:564036", 10, 10, 28, 91, 2588687752, 1168371662, 1623033341, 1465362298, 564036, "SRX23412817", "SRS20268138", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30267, "SRR27747517", "SRX23412817", "SRS20268138", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 3", "GSM8038039", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 3", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a", "GSM8038039", "GSM8038039: All vestibular neurons  replicate 3; Danio rerio; RNA Seq", "GSM8038039 r1", "GSM8038039", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Zebrafish-Replicate3_S3_L004_I1_001.fastq.gz Zebrafish-Replicate3_S3_L004_I2_001.fastq.gz Zebrafish-Replicate3_S3_L004_R1_001.fastq.gz Zebrafish-Replicate3_S3_L004_R2_001.fastq.gz", "fastq fastq fastq fastq", 10455949462.0, 75222658.0, "GSM8038039 r4", "0:10 1:10 2:28 3:91", "A:2581792541;C:1170458560;G:1624194611;T:1468248274;N:567892", 10, 10, 28, 91, 2581792541, 1170458560, 1624194611, 1468248274, 567892, "SRX23412817", "SRS20268138", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30268, "SRR27747518", "SRX23412816", "SRS20268137", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 2", "GSM8038038", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. One replicate was photoconverted at 36 hpf to isolate early born red  converted Kaede from late born green  unconverted Kaede neurons.", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a", "GSM8038038", "GSM8038038: All vestibular neurons  replicate 2; Danio rerio; RNA Seq", "GSM8038038 r1", "GSM8038038", "1", "Harvested neurons were sorted to isolate single cells  resuspended in L15+2% FBS  and kept on ice until cDNA synthesis Performed using manufacturer's instructions; 10x Genomics  three prime V3.1", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP486180", null, "loader:fastq load.py", "Zebrafish-Replicate2_S2_L001_I1_001.fastq.gz Zebrafish-Replicate2_S2_L001_I2_001.fastq.gz Zebrafish-Replicate2_S2_L001_R1_001.fastq.gz Zebrafish-Replicate2_S2_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 9414131535.0, 67727565.0, "GSM8038038 r1", "0:10 1:10 2:28 3:91", "A:2659651693;C:894536960;G:1598721159;T:1009819179;N:479424", 10, 10, 28, 91, 2659651693, 894536960, 1598721159, 1009819179, 479424, "SRX23412816", "SRS20268137", "SRA1792542", "Neuroscience Institute, New York University Grossman School of Medicine", "Neuroscience Institute, New York University Grossman School of Medicine", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-01-26", "Multi-stage", "Multi-stage", "Brain", "Nervous System"], [30269, "SRR27747519", "SRX23412816", "SRS20268137", "SRP486180", "PRJNA1069776", "Molecular characterization of hindbrain vestibular neurons in the larval zebrafish scRNA Seq", "GSE254346", "Transcriptome Analysis", "The molecular logic that specifies and assembles closely related subtypes of neurons into functional sensorimotor circuits remains unclear. The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. One sample had two conditions: neurons born before or post a previously identified \"midpoint\" in hindbrain vestibular neuron development 36 hpf  labeled using Kaede photoconversions. Neurons were sequenced with 10x Genomics.", null, null, null, "All vestibular neurons  replicate 2", "GSM8038038", null, "tissue:Hindbrain vestibular neurons|time:72 hpf 74 hpf type:Hindbrain vestibular neurons|genotype:Tg 6.7Tru.Hcrtr2:GAL4 VP16; TgUAS:E1b Kaede|treatment:n/a|geo loc name:missing|collection date:missing", "All vestibular neurons  replicate 2", "Cell Ranger v7.0.0 Assembly: GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "Hindbrain vestibular neurons", "Three replicates were untreated. 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The goal of this study was to characterize the molecular profiles of hindbrain vestibular neurons in the larval zebrafish to identify candidate molecular programs that specify their subtype fate  topography  and circuit assembly. We used single cell RNA sequencing to generate a comprehensive atlas of hindbrain vestibular neurons and fluorecent in situ hybridization to annotate profiled neurons. Our dataset serves as a reference for evaluating developmental changes in molecular profiles following perturbations and identifies new candidate molecular solutions that assemble closely related subtypes into functional circuits. Overall design: Hindbrain vestibular neurons  labeled by Tg 6.7Tru.Hcrtr2:GAL4 VP16;TgUAS:E1b Kaede  harvested from zebrafish embryos between 72 hpf 74 hpf. Fluorescent neurons were isolated by fluorescence activated cell sorting FACS according to Kaede fluorescence. Three samples contained all hindbrain vestibular neurons sorted. 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