{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where devstage_curation = \"Multi-stage\" and experiment.library_source = \"TRANSCRIPTOMIC SINGLE CELL\"", "rows": [[9898, "ERR4194114", "ERX4155254", "ERS4601292", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. 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Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. 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For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. 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Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 1", "SAMEA6873728", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873728|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 1|age:6|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 1|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 1 s", "Sample 1 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:6|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA6h_1_S1_L001.bam", "bam", 7181772357.0, 71106657.0, "E MTAB 9193:cDNA6h 1 S1 L001", "0:101", "A:2074032710;C:1415439071;G:1544274166;T:2140112533;N:7913877", 101, null, null, null, 2074032710, 1415439071, 1544274166, 2140112533, 7913877, "ERX4155253", "ERS4601291", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.9297, null, 0.12156, null, 0.8117, null, 0.51457, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9901, "ERR4194113", "ERX4155253", "ERS4601291", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 1", "SAMEA6873728", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873728|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 1|age:6|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 1|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 1 s", "Sample 1 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:6|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA6h_1_S1_L002.bam", "bam", 7256542253.0, 71846953.0, "E MTAB 9193:cDNA6h 1 S1 L002", "0:101", "A:2096163385;C:1430325700;G:1560530043;T:2162265616;N:7257509", 101, null, null, null, 2096163385, 1430325700, 1560530043, 2162265616, 7257509, "ERX4155253", "ERS4601291", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.92882, null, 0.12127, null, 0.81162, null, 0.51457, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9902, "ERR4194128", "ERX4155256", "ERS4601294", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 4", "SAMEA6873731", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873731|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 4|age:13|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 4|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 4 s", "Sample 4 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:13|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA13h_1_control_S1_L001.bam", "bam", 10785974123.0, 106791823.0, "E MTAB 9193:cDNA13h 1 control S1 L001", "0:101", "A:3007305661;C:2241057062;G:2505494392;T:2986021199;N:46095809", 101, null, null, null, 3007305661, 2241057062, 2505494392, 2986021199, 46095809, "ERX4155256", "ERS4601294", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.67094, null, 0.07571, null, 0.92951, null, 0.5272, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9903, "ERR4194129", "ERX4155256", "ERS4601294", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 4", "SAMEA6873731", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873731|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 4|age:13|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 4|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 4 s", "Sample 4 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:13|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA13h_1_control_S1_L002.bam", "bam", 10595192294.0, 104902894.0, "E MTAB 9193:cDNA13h 1 control S1 L002", "0:101", "A:2971916419;C:2204229263;G:2390854035;T:2949877754;N:78314823", 101, null, null, null, 2971916419, 2204229263, 2390854035, 2949877754, 78314823, "ERX4155256", "ERS4601294", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.67255, null, 0.08054, null, 0.91583, null, 0.52661, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9904, "ERR4194116", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_1_S3_L001.bam", "bam", 1622536412.0, 16556494.0, "E MTAB 9193:cDNA10h 1 S3 L001", "0:98", "A:491141175;C:317161060;G:354179755;T:459976409;N:78013", 98, null, null, null, 491141175, 317161060, 354179755, 459976409, 78013, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93238, null, 0.13033, null, 0.89132, null, 0.47076, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9905, "ERR4194117", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_1_S3_L002.bam", "bam", 1484328776.0, 15146212.0, "E MTAB 9193:cDNA10h 1 S3 L002", "0:98", "A:450782055;C:290088574;G:322801188;T:420570801;N:86158", 98, null, null, null, 450782055, 290088574, 322801188, 420570801, 86158, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.92846, null, 0.13128, null, 0.89923, null, 0.46879, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9906, "ERR4194118", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_1_S3_L003.bam", "bam", 1578833116.0, 16110542.0, "E MTAB 9193:cDNA10h 1 S3 L003", "0:98", "A:477600666;C:308260969;G:347303289;T:445467384;N:200808", 98, null, null, null, 477600666, 308260969, 347303289, 445467384, 200808, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93107, null, 0.12965, null, 0.91265, null, 0.47375, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9907, "ERR4194119", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_1_S3_L004.bam", "bam", 1859564994.0, 18975153.0, "E MTAB 9193:cDNA10h 1 S3 L004", "0:98", "A:562936790;C:364057256;G:406607636;T:525811595;N:151717", 98, null, null, null, 562936790, 364057256, 406607636, 525811595, 151717, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93453, null, 0.1266, null, 0.87714, null, 0.46957, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9908, "ERR4194120", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L001.bam", "bam", 1156341102.0, 11799399.0, "E MTAB 9193:cDNA10h 2 S3 L001", "0:98", "A:345857612;C:224453195;G:257551986;T:327969064;N:509245", 98, null, null, null, 345857612, 224453195, 257551986, 327969064, 509245, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93863, null, 0.10744, null, 0.81984, null, 0.501, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9909, "ERR4194121", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L002.bam", "bam", 1105219696.0, 11277752.0, "E MTAB 9193:cDNA10h 2 S3 L002", "0:98", "A:331051973;C:214369903;G:246546338;T:312897773;N:353709", 98, null, null, null, 331051973, 214369903, 246546338, 312897773, 353709, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93941, null, 0.10818, null, 0.82211, null, 0.50079, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9910, "ERR4194122", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L003.bam", "bam", 1105199606.0, 11277547.0, "E MTAB 9193:cDNA10h 2 S3 L003", "0:98", "A:331158619;C:214305131;G:246465559;T:312930408;N:339889", 98, null, null, null, 331158619, 214305131, 246465559, 312930408, 339889, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93767, null, 0.10645, null, 0.82329, null, 0.50057, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9911, "ERR4194123", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L004.bam", "bam", 1108707712.0, 11313344.0, "E MTAB 9193:cDNA10h 2 S3 L004", "0:98", "A:331374433;C:215925150;G:247131942;T:313865174;N:411013", 98, null, null, null, 331374433, 215925150, 247131942, 313865174, 411013, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93827, null, 0.10842, null, 0.82031, null, 0.49241, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9912, "ERR4194124", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L005.bam", "bam", 1110084612.0, 11327394.0, "E MTAB 9193:cDNA10h 2 S3 L005", "0:98", "A:332881960;C:215360330;G:247563319;T:313842331;N:436672", 98, null, null, null, 332881960, 215360330, 247563319, 313842331, 436672, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93827, null, 0.10719, null, 0.8238, null, 0.50406, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9913, "ERR4194125", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L006.bam", "bam", 1113812728.0, 11365436.0, "E MTAB 9193:cDNA10h 2 S3 L006", "0:98", "A:333112288;C:216649152;G:248341020;T:315338848;N:371420", 98, null, null, null, 333112288, 216649152, 248341020, 315338848, 371420, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93843, null, 0.10675, null, 0.82079, null, 0.49284, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9914, "ERR4194126", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L007.bam", "bam", 1119495748.0, 11423426.0, "E MTAB 9193:cDNA10h 2 S3 L007", "0:98", "A:335288339;C:217283719;G:249624836;T:316900388;N:398466", 98, null, null, null, 335288339, 217283719, 249624836, 316900388, 398466, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93711, null, 0.10749, null, 0.82266, null, 0.49382, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9915, "ERR4194127", "ERX4155255", "ERS4601293", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 3", "SAMEA6873730", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873730|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 3|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 3 s", "Sample 3 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:10|Experimental Factor: RNA interference:n1", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA10h_2_S3_L008.bam", "bam", 1175946688.0, 11999456.0, "E MTAB 9193:cDNA10h 2 S3 L008", "0:98", "A:351364169;C:228318193;G:261971072;T:333863943;N:429311", 98, null, null, null, 351364169, 228318193, 261971072, 333863943, 429311, "ERX4155255", "ERS4601293", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.93851, null, 0.10755, null, 0.82158, null, 0.49895, null, 98, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9916, "ERR4194130", "ERX4155257", "ERS4601295", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 5", "SAMEA6873732", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873732|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 5|age:13|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 5|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 5 s", "Sample 5 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:13|Experimental Factor: RNA interference:morpholino mediated Gata5/6 knockdown", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA13h_2_gata56KD_S2_L001.bam", "bam", 7791309377.0, 77141677.0, "E MTAB 9193:cDNA13h 2 gata56KD S2 L001", "0:101", "A:2198242220;C:1593746618;G:1784167452;T:2181979820;N:33173267", 101, null, null, null, 2198242220, 1593746618, 1784167452, 2181979820, 33173267, "ERX4155257", "ERS4601295", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.66736, null, 0.08039, null, 0.93026, null, 0.50796, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [9917, "ERR4194131", "ERX4155257", "ERS4601295", "ERP122151", "PRJEB38705", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E-MTAB-9193", "Transcriptome Analysis", "TgBACgata5:EGFP transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6 8 10 13 hpf and morpholinos injected Gata5/6 know down KD embryos at 13 hpf were pre screened for GFP signal and dissociated into single cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting FACS. Single cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform Rapid Run Model. Note that the 10h single cell library was sequenced twice Named as cDNA10h 1* and cDNA10h 2* separately.", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", null, "Protocols: WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200 \u03bcl calcium free Ringer solution 116 mM NaCl 2.6 mM KCl 5 mM HEPE pH 7.0 for 5 min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100 \u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Sample 5", "SAMEA6873732", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada", "ENA FIRST PUBLIC:2020 12 31T11:11:47Z|ENA LAST UPDATE:2020 06 04T16:16:46Z|External Id:SAMEA6873732|INSDC center name:Developmental and Stem Cell Biology Sickkids Toronto Canada; Genetics and Genome Biology Sickkids Toronto Canada; Molecular Genetics University of Toronto Toronto Canada|INSDC first public:2020 12 31T11:11:47Z|INSDC last update:2020 06 04T16:16:46Z|INSDC status:public|Submitter Id:E MTAB 9193:Sample 5|age:13|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:TgBACgata5: EGFPpd25|immunophenotype:GFP+|individual:embryo pool 100 200|organism part:mesendoderm|sample name:E MTAB 9193:Sample 5|scientific name:Danio rerio|sex:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "E MTAB 9193:Sample 5 s", "Sample 5 s", "Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "WT and Gata5/6KD TgBAC gata5: EGFP embryos were synchronized and dechorionated with pronase Sigma Cat# 11459643001 at desired stages. For embryos at gastrulation stages 6 hpf 8 hpf 10 hpf the dissociation was performed by incubating embryos in 200\u2009\u03bcl calcium free Ringer solution 116\u2009mM NaCl 2.6\u2009mM KCl 5\u2009mM HEPE pH 7.0 for 5\u2009min followed by 500ul TrpLE GIBCO TrypLE Express Enzyme cat #: 12604 013 at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single cell suspensions were generated. Embryos at the early segmentation stage 13 hpf were treated similarly but incubated with 500 ul of 0.25 Trypsin GIBCO Trypsin2.5% no phenol red cat#: 15090046/ EDTA instead of TrpLE for dissociation. post dissociation Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence activated cell sorting FACS. FACS was performed on Sony SH800S Cell Sorter MoFlo XDP or MoFlo Astrios with a 100\u2009\u03bcm nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Around 20 000 to 50 000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions post FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell three prime Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.", "Experimental Factor: age:13|Experimental Factor: RNA interference:morpholino mediated Gata5/6 knockdown", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "PolyA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2500", null, "ERP122151", "Illumina HiSeq 2500 sequencing; Single cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish", "ENA FIRST PUBLIC:2021 06 02|ENA LAST UPDATE:2021 12 01", "cDNA13h_2_gata56KD_S2_L002.bam", "bam", 7658149967.0, 75823267.0, "E MTAB 9193:cDNA13h 2 gata56KD S2 L002", "0:101", "A:2172230233;C:1568805100;G:1703587704;T:2157181071;N:56345859", 101, null, null, null, 2172230233, 1568805100, 1703587704, 2157181071, 56345859, "ERX4155257", "ERS4601295", "ERA2667966", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", "Developmental and Stem Cell Biology, Sickkids, Toronto, Canada; Genetics and Genome Biology, Sickkids, Toronto, Canada; Molecular Genetics, University of Toronto, Toronto, Canada|European Nucleotide Archive", 1, 0.66565, null, 0.08597, null, 0.91804, null, 0.51772, null, 101, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "Canada", "2020-06-04", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [11130, "ERR9979395", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L002_R1_001.fastq.gz P22202_7002_S2_L002_R2_001.fastq.gz", "fastq fastq", 18144406998.0, 153766161.0, "E MTAB 11984:P22202 7002 S2 L002", "0:28 1:90", "A:5277424121;C:3880928155;G:4118706097;T:4866649878;N:698747", 28, 90, null, null, 5277424121, 3880928155, 4118706097, 4866649878, 698747, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.0109, 0.90145, 0.0053, 0.21373, 0.98746, 0.79423, 0.41198, 0.54447, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11131, "ERR9979394", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L001_R1_001.fastq.gz P22202_7002_S2_L001_R2_001.fastq.gz", "fastq fastq", 18458951574.0, 156431793.0, "E MTAB 11984:P22202 7002 S2 L001", "0:28 1:90", "A:5372920926;C:3946687814;G:4185647697;T:4953205454;N:489683", 28, 90, null, null, 5372920926, 3946687814, 4185647697, 4953205454, 489683, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01037, 0.90168, 0.0048, 0.21246, 0.98752, 0.79297, 0.42601, 0.54996, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11132, "ERR9979392", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L001_R1_001.fastq.gz P22202_7001_S1_L001_R2_001.fastq.gz", "fastq fastq", 19396994226.0, 164381307.0, "E MTAB 11984:P22202 7001 S1 L001", "0:28 1:90", "A:5665199484;C:4091196594;G:4415211076;T:5224866754;N:520318", 28, 90, null, null, 5665199484, 4091196594, 4415211076, 5224866754, 520318, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01093, 0.89238, 0.00557, 0.232, 0.98764, 0.79444, 0.40806, 0.54781, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11133, "ERR9979393", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L002_R1_001.fastq.gz P22202_7001_S1_L002_R2_001.fastq.gz", "fastq fastq", 19063958224.0, 161558968.0, "E MTAB 11984:P22202 7001 S1 L002", "0:28 1:90", "A:5563588848;C:4022930643;G:4344549562;T:5132136743;N:752428", 28, 90, null, null, 5563588848, 4022930643, 4344549562, 5132136743, 752428, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01118, 0.89308, 0.00573, 0.23128, 0.98737, 0.79354, 0.39264, 0.54552, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11238, "ERR10782555", "ERX10233132", "ERS14439197", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 6", "E MTAB 12503:Sample 6", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 6|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 6 p", "Sample 6 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-6_R1.fastq.gz 20170530.A-6_R2.fastq.gz", "fastq fastq", 14809693138.0, 49038719.0, "E MTAB 12503:20170530.A 6 R", "0:151 1:151", "A:4069749497;C:3367864217;G:3430269434;T:3928390721;N:13419269", 151, 151, null, null, 4069749497, 3367864217, 3430269434, 3928390721, 13419269, "ERX10233132", "ERS14439197", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.83945, 0.69977, 0.27269, 0.22488, 0.74523, 0.77654, 0.4899, 0.4358, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11239, "ERR10782554", "ERX10233131", "ERS14439196", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 5", "E MTAB 12503:Sample 5", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 5|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 5 p", "Sample 5 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-5_R1.fastq.gz 20170530.A-5_R2.fastq.gz", "fastq fastq", 14954968728.0, 49519764.0, "E MTAB 12503:20170530.A 5 R", "0:151 1:151", "A:4063340262;C:3440933120;G:3455723079;T:3981396987;N:13575280", 151, 151, null, null, 4063340262, 3440933120, 3455723079, 3981396987, 13575280, "ERX10233131", "ERS14439196", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.85891, 0.86859, 0.28842, 0.28866, 0.7349, 0.75051, 0.45532, 0.49145, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11242, "ERR10782551", "ERX10233128", "ERS14439193", "ERP144048", "PRJEB58983", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E-MTAB-12503", "Transcriptome Analysis", "Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, "Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Sample 7", "E MTAB 12503:Sample 7", null, "strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 7|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:rbm8a d5/d5|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", null, null, null, null, null, null, null, null, "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "E MTAB 12503:Sample 7 p", "Sample 7 p", "RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer\u00e2\u20ac\u2122s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.", "Experimental Factor: developmental stage:bud|Experimental Factor: genotype:rbm8a d5/d5", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "ERP144048", "Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling", "ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31", "20170530.A-7_R1.fastq.gz 20170530.A-7_R2.fastq.gz", "fastq fastq", 12726557236.0, 42140918.0, "E MTAB 12503:20170530.A 7 R", "0:151 1:151", "A:3510176970;C:2879679978;G:2932244172;T:3392911734;N:11544382", 151, 151, null, null, 3510176970, 2879679978, 2932244172, 3392911734, 11544382, "ERX10233128", "ERS14439193", "ERA20162442", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 2, 0.88531, 0.88678, 0.33279, 0.3329, 0.73545, 0.74992, 0.52548, 0.52864, 151, 151, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "sc", "unknown", "unknown", null, "Switzerland", "2023-03-31", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [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"], [29175, "SRR27292304", "SRX22969963", "SRS19936063", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 48 hours post burn", "GSM7982886", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 48 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982886", "GSM7982886: Burned Larval Zebrafish 48 hours post burn; Danio rerio; RNA Seq", "GSM7982886 r1", "GSM7982886", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-48hpb_S5_L001_R2_001.fastq.gz Burn-48hpb_S5_L001_R1_001.fastq.gz", "fastq fastq", 20874980529.0, 178418637.0, "GSM7982886 r1", "0:28 1:89", "A:5945091269;C:4392590896;G:4497688824;T:6035946283;N:3663257", 28, 89, null, null, 5945091269, 4392590896, 4497688824, 6035946283, 3663257, "SRX22969963", "SRS19936063", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00573, 0.91666, 0.00222, 0.19474, 0.9931, 0.79306, 0.42082, 0.53801, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29176, "SRR27292305", "SRX22969963", "SRS19936063", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 48 hours post burn", "GSM7982886", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 48 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982886", "GSM7982886: Burned Larval Zebrafish 48 hours post burn; Danio rerio; RNA Seq", "GSM7982886 r1", "GSM7982886", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-48hpb_S5_L002_R1_001.fastq.gz Burn-48hpb_S5_L002_R2_001.fastq.gz", "fastq fastq", 20022973425.0, 171136525.0, "GSM7982886 r2", "0:28 1:89", "A:5709681181;C:4208386174;G:4307510275;T:5796282368;N:1113427", 28, 89, null, null, 5709681181, 4208386174, 4307510275, 5796282368, 1113427, "SRX22969963", "SRS19936063", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00547, 0.9154, 0.00202, 0.1955, 0.99283, 0.79423, 0.39307, 0.53427, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29177, "SRR27292306", "SRX22969962", "SRS19936064", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 24 hours post burn", "GSM7982885", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 24 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982885", "GSM7982885: Burned Larval Zebrafish 24 hours post burn; Danio rerio; RNA Seq", "GSM7982885 r1", "GSM7982885", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-24hpb_S2_L001_R1_001.fastq.gz Burn-24hpb_S2_L001_R2_001.fastq.gz", "fastq fastq", 9782870058.0, 83614274.0, "GSM7982885 r1", "0:28 1:89", "A:2744737668;C:2140848416;G:2249400157;T:2645170677;N:2713140", 28, 89, null, null, 2744737668, 2140848416, 2249400157, 2645170677, 2713140, "SRX22969962", "SRS19936064", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00752, 0.92059, 0.00244, 0.16143, 0.98944, 0.82929, 0.44176, 0.54525, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29178, "SRR27292307", "SRX22969962", "SRS19936064", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 24 hours post burn", "GSM7982885", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 24 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982885", "GSM7982885: Burned Larval Zebrafish 24 hours post burn; Danio rerio; RNA Seq", "GSM7982885 r1", "GSM7982885", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-24hpb_S2_L002_R2_001.fastq.gz Burn-24hpb_S2_L002_R1_001.fastq.gz", "fastq fastq", 9861989202.0, 84290506.0, "GSM7982885 r2", "0:28 1:89", "A:2769268861;C:2158644227;G:2263475493;T:2669311436;N:1289185", 28, 89, null, null, 2769268861, 2158644227, 2263475493, 2669311436, 1289185, "SRX22969962", "SRS19936064", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.0076, 0.9197, 0.00232, 0.16162, 0.98995, 0.82852, 0.43786, 0.55256, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29179, "SRR27292308", "SRX22969961", "SRS19936062", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 6 hours post burn", "GSM7982884", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 6 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982884", "GSM7982884: Burned Larval Zebrafish 6 hours post burn; Danio rerio; RNA Seq", "GSM7982884 r1", "GSM7982884", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-6hpb_S7_L001_R1_001.fastq.gz Burn-6hpb_S7_L001_R2_001.fastq.gz", "fastq fastq", 12475938384.0, 106631952.0, "GSM7982884 r1", "0:28 1:89", "A:3401989108;C:2878275107;G:3114945415;T:3078525361;N:2203393", 28, 89, null, null, 3401989108, 2878275107, 3114945415, 3078525361, 2203393, "SRX22969961", "SRS19936062", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.01053, 0.89396, 0.00429, 0.18315, 0.99458, 0.85756, 0.34876, 0.58728, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29180, "SRR27292309", "SRX22969961", "SRS19936062", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Burned Larval Zebrafish 6 hours post burn", "GSM7982884", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing", "Burned Larval Zebrafish 6 hours post burn", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn", "GSM7982884", "GSM7982884: Burned Larval Zebrafish 6 hours post burn; Danio rerio; RNA Seq", "GSM7982884 r1", "GSM7982884", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "Burn-6hpb_S7_L002_R1_001.fastq.gz Burn-6hpb_S7_L002_R2_001.fastq.gz", "fastq fastq", 11874083247.0, 101487891.0, "GSM7982884 r2", "0:28 1:89", "A:3243501055;C:2735412615;G:2958387721;T:2936117380;N:664476", 28, 89, null, null, 3243501055, 2735412615, 2958387721, 2936117380, 664476, "SRX22969961", "SRS19936062", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.01063, 0.89389, 0.00439, 0.18047, 0.99403, 0.85878, 0.36393, 0.54831, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29181, "SRR27292310", "SRX22969960", "SRS19936061", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 5 dpf", "GSM7982883", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 5 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982883", "GSM7982883: Unwounded Larval Zebrafish 5 dpf Danio rerio; RNA Seq", "GSM7982883 r1", "GSM7982883", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-5dpf_S6_L001_R2_001.fastq.gz WT-5dpf_S6_L001_R1_001.fastq.gz", "fastq fastq", 31813158312.0, 271907336.0, "GSM7982883 r1", "0:28 1:89", "A:8991313503;C:6911976524;G:7105775004;T:8798521232;N:5572049", 28, 89, null, null, 8991313503, 6911976524, 7105775004, 8798521232, 5572049, "SRX22969960", "SRS19936061", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00661, 0.92343, 0.00245, 0.19293, 0.99255, 0.81379, 0.35935, 0.54818, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29182, "SRR27292311", "SRX22969960", "SRS19936061", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 5 dpf", "GSM7982883", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 5 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982883", "GSM7982883: Unwounded Larval Zebrafish 5 dpf Danio rerio; RNA Seq", "GSM7982883 r1", "GSM7982883", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-5dpf_S6_L002_R1_001.fastq.gz WT-5dpf_S6_L002_R2_001.fastq.gz", "fastq fastq", 30524386113.0, 260892189.0, "GSM7982883 r2", "0:28 1:89", "A:8637605077;C:6625244702;G:6807369409;T:8452468968;N:1697957", 28, 89, null, null, 8637605077, 6625244702, 6807369409, 8452468968, 1697957, "SRX22969960", "SRS19936061", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00648, 0.92418, 0.00233, 0.19549, 0.99241, 0.81444, 0.37733, 0.54674, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29183, "SRR27292312", "SRX22969959", "SRS19936060", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 4 dpf", "GSM7982882", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 4 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982882", "GSM7982882: Unwounded Larval Zebrafish 4 dpf Danio rerio; RNA Seq", "GSM7982882 r1", "GSM7982882", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-4dpf_S1_L001_R1_001.fastq.gz WT-4dpf_S1_L001_R2_001.fastq.gz", "fastq fastq", 12682286721.0, 108395613.0, "GSM7982882 r1", "0:28 1:89", "A:3575375325;C:2777398557;G:2900088646;T:3425909511;N:3514682", 28, 89, null, null, 3575375325, 2777398557, 2900088646, 3425909511, 3514682, "SRX22969959", "SRS19936060", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00505, 0.92112, 0.00185, 0.16865, 0.99257, 0.8258, 0.38216, 0.5512, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29184, "SRR27292313", "SRX22969959", "SRS19936060", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 4 dpf", "GSM7982882", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 4 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982882", "GSM7982882: Unwounded Larval Zebrafish 4 dpf Danio rerio; RNA Seq", "GSM7982882 r1", "GSM7982882", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-4dpf_S1_L002_R2_001.fastq.gz WT-4dpf_S1_L002_R1_001.fastq.gz", "fastq fastq", 12832453413.0, 109679089.0, "GSM7982882 r2", "0:28 1:89", "A:3620789787;C:2810897812;G:2929508681;T:3469577031;N:1680102", 28, 89, null, null, 3620789787, 2810897812, 2929508681, 3469577031, 1680102, "SRX22969959", "SRS19936060", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.0052, 0.92355, 0.00192, 0.16775, 0.99263, 0.8253, 0.37089, 0.55115, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29185, "SRR27292314", "SRX22969958", "SRS19936059", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 3 dpf", "GSM7982881", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 3 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982881", "GSM7982881: Unwounded Larval Zebrafish 3 dpf Danio rerio; RNA Seq", "GSM7982881 r1", "GSM7982881", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-3dpf_S4_L001_R1_001.fastq.gz WT-3dpf_S4_L001_R2_001.fastq.gz", "fastq fastq", 7586138430.0, 64838790.0, "GSM7982881 r1", "0:28 1:89", "A:2101749534;C:1692875352;G:1784872358;T:2005308370;N:1332816", 28, 89, null, null, 2101749534, 1692875352, 1784872358, 2005308370, 1332816, "SRX22969958", "SRS19936059", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00976, 0.92096, 0.00368, 0.18077, 0.99356, 0.83875, 0.3661, 0.59073, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic System"], [29186, "SRR27292315", "SRX22969958", "SRS19936059", "SRP479046", "PRJNA1054617", "Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish", "GSE250610", "Transcriptome Analysis", "Neutrophils accumulate early in burn wounds and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways we found il6 il6r signaling as a time point specific macrophage neutrophil interaction suggesting the importance of timing in innate immune response in burn. Finally to test the translational value of our fish model we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222 200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin 1mM EDTA in PBS at 28.5\u00b0C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2 100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 \u00b0C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris singles versus doublets live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.", null, "pubmed:38922186", null, "Unwounded Larval Zebrafish 3 dpf", "GSM7982881", null, "source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing", "Unwounded Larval Zebrafish 3 dpf", "FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days Burn 24 hours v6.1.2 Unwounded 3days Unwounded 5days Burn 6hours Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix", "Whole blood", "Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", "Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5\u02daC incubator.", "tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded", "GSM7982881", "GSM7982881: Unwounded Larval Zebrafish 3 dpf Danio rerio; RNA Seq", "GSM7982881 r1", "GSM7982881", "1", "Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP479046", null, "loader:fastq load.py", "WT-3dpf_S4_L002_R1_001.fastq.gz WT-3dpf_S4_L002_R2_001.fastq.gz", "fastq fastq", 7258692168.0, 62040104.0, "GSM7982881 r2", "0:28 1:89", "A:2013529524;C:1617959197;G:1704796898;T:1922004726;N:401823", 28, 89, null, null, 2013529524, 1617959197, 1704796898, 1922004726, 401823, "SRX22969958", "SRS19936059", "SRA1772557", "University of Wisconsin-Madison", "University of Wisconsin-Madison", 2, 0.00949, 0.92207, 0.00359, 0.18081, 0.99375, 0.83751, 0.37347, 0.55889, 28, 89, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "random_priming", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-12-19", "Multi-stage", "Multi-stage", "Blood", "Hematopoietic 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. 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_L002_I1_001.fastq.gz Zebrafish-Replicate2_S2_L002_I2_001.fastq.gz Zebrafish-Replicate2_S2_L002_R1_001.fastq.gz Zebrafish-Replicate2_S2_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 9495760675.0, 68314825.0, "GSM8038038 r2", "0:10 1:10 2:28 3:91", "A:2685940540;C:901627244;G:1612080998;T:1016532106;N:468187", 10, 10, 28, 91, 2685940540, 901627244, 1612080998, 1016532106, 468187, "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"], [30270, "SRR27747520", "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_L003_I1_001.fastq.gz Zebrafish-Replicate2_S2_L003_I2_001.fastq.gz Zebrafish-Replicate2_S2_L003_R1_001.fastq.gz Zebrafish-Replicate2_S2_L003_R2_001.fastq.gz", "fastq fastq fastq fastq", 9299053574.0, 66899666.0, "GSM8038038 r3", "0:10 1:10 2:28 3:91", "A:2619668178;C:887003175;G:1580306677;T:1000391033;N:500543", 10, 10, 28, 91, 2619668178, 887003175, 1580306677, 1000391033, 500543, "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"], [30271, "SRR27747521", "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_L004_I1_001.fastq.gz Zebrafish-Replicate2_S2_L004_I2_001.fastq.gz Zebrafish-Replicate2_S2_L004_R1_001.fastq.gz Zebrafish-Replicate2_S2_L004_R2_001.fastq.gz", "fastq fastq fastq fastq", 9269439763.0, 66686617.0, "GSM8038038 r4", "0:10 1:10 2:28 3:91", "A:2605497678;C:885933483;G:1577360287;T:999190994;N:499705", 10, 10, 28, 91, 2605497678, 885933483, 1577360287, 999190994, 499705, "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"], [30272, "SRR27747522", "SRX23412815", "SRS20268136", "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 1", "GSM8038037", 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 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:n/a", "GSM8038037", "GSM8038037: All vestibular neurons replicate 1; Danio rerio; RNA Seq", "GSM8038037 r1", "GSM8038037", "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-Replicate1_S1_L001_I1_001.fastq.gz Zebrafish-Replicate1_S1_L001_I2_001.fastq.gz Zebrafish-Replicate1_S1_L001_R1_001.fastq.gz Zebrafish-Replicate1_S1_L001_R2_001.fastq.gz", "fastq fastq fastq fastq", 11844241708.0, 85210372.0, "GSM8038037 r1", "0:10 1:10 2:28 3:91", "A:3114181870;C:1253864519;G:2080987238;T:1304504811;N:605414", 10, 10, 28, 91, 3114181870, 1253864519, 2080987238, 1304504811, 605414, "SRX23412815", "SRS20268136", "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"], [30273, "SRR27747523", "SRX23412815", "SRS20268136", "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 1", "GSM8038037", 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 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:n/a", "GSM8038037", "GSM8038037: All vestibular neurons replicate 1; Danio rerio; RNA Seq", "GSM8038037 r1", "GSM8038037", "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-Replicate1_S1_L002_I1_001.fastq.gz Zebrafish-Replicate1_S1_L002_I2_001.fastq.gz Zebrafish-Replicate1_S1_L002_R1_001.fastq.gz Zebrafish-Replicate1_S1_L002_R2_001.fastq.gz", "fastq fastq fastq fastq", 11897506230.0, 85593570.0, "GSM8038037 r2", "0:10 1:10 2:28 3:91", "A:3132719148;C:1257962906;G:2089627351;T:1308117927;N:587538", 10, 10, 28, 91, 3132719148, 1257962906, 2089627351, 1308117927, 587538, "SRX23412815", "SRS20268136", "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"], [30274, "SRR27747524", "SRX23412815", "SRS20268136", "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 1", "GSM8038037", 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 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:n/a", "GSM8038037", "GSM8038037: All vestibular neurons replicate 1; Danio rerio; RNA Seq", "GSM8038037 r1", "GSM8038037", "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-Replicate1_S1_L003_I1_001.fastq.gz Zebrafish-Replicate1_S1_L003_I2_001.fastq.gz Zebrafish-Replicate1_S1_L003_R1_001.fastq.gz Zebrafish-Replicate1_S1_L003_R2_001.fastq.gz", "fastq fastq fastq fastq", 11710536913.0, 84248467.0, "GSM8038037 r3", "0:10 1:10 2:28 3:91", "A:3071431897;C:1243257065;G:2058561915;T:1292732765;N:626855", 10, 10, 28, 91, 3071431897, 1243257065, 2058561915, 1292732765, 626855, "SRX23412815", "SRS20268136", "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"], [30275, "SRR27747525", "SRX23412815", "SRS20268136", "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 1", "GSM8038037", 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 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:n/a", "GSM8038037", "GSM8038037: All vestibular neurons replicate 1; Danio rerio; RNA Seq", "GSM8038037 r1", "GSM8038037", "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-Replicate1_S1_L004_I1_001.fastq.gz Zebrafish-Replicate1_S1_L004_I2_001.fastq.gz Zebrafish-Replicate1_S1_L004_R1_001.fastq.gz Zebrafish-Replicate1_S1_L004_R2_001.fastq.gz", "fastq fastq fastq fastq", 11691727294.0, 84113146.0, "GSM8038037 r4", "0:10 1:10 2:28 3:91", "A:3060624267;C:1243160034;G:2057238308;T:1292649886;N:623791", 10, 10, 28, 91, 3060624267, 1243160034, 2057238308, 1292649886, 623791, "SRX23412815", "SRS20268136", "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"], [30662, "SRR28125659", "SRX23770350", "SRS20595835", "SRP492124", "PRJNA1081449", "Thymic mimetic cells in zebrafish", "GSE259418", "Transcriptome Analysis", "We profiled the zebrafish thymus by single cell RNA sequencing to investigate the heterogeneity of zebrafish thymic epithelial cells and determine whether similar mimetic cell populations to those found in mice and humans might also exist in the zebrafish. Overall design: To assess the heterogeneity of zebrafish thymic epithelial cells a total of 11 zebrafish samples derived from 14 adult and 21 juvenile zebrafish were studied. Specifically we re processed single cell RNA sequencing scRNA seq data from 8 zebrafish thymus samples derived from 4 adult zebrafish and 21 juvenile zebrafish previously described in GSE190794 GSM5732067 GSM5732070 GSM5732077 GSM5732078 GSM5732079 GSM5732080 GSM5732081 GSM5732082 so that reads mapping to intronic regions were included in the analysis to increase sensitivity and be consistent with mouse and human scRNA seq studies for cross species comparison. In addition we generated 3 new scRNA seq datasets derived from a total of 10 adult lck:mCherry;cd79a:eGFP transgenic zebrafish. The thymi from 5 of these adult zebrafish were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII whereas thymi from the other 5 adult zebrafish underwent enzymatic dissociation with Liberase TM prior to quenching filtering and cell sorting. The live non autofluorescent GFP negative mCherry negative cells a population depleted of T and B cells thus providing a relative enrichment of thymic epithelial cells were sorted and collected for 10x scRNA seq. One 10x lane was used for the mechanically dissociated sample untreated and two lanes were used for the liberase treated sample to obtain 2 technical replicates. Re analysed Samples in GSE190794/PRJNA788489 and their processed data file: SampleName Experiment BioSample processed data file GSM5732067 Adult Thymus 3 SRX13397695 SAMN24007722 Fish 22 Thymus .filtered feature bc matrix.h5 GSM5732070 Adult Thymus 4 SRX13397698 SAMN24007725 Fish 24 Thymus .filtered feature bc matrix.h5 GSM5732077 Adult Thymus 1 SRX13397705 SAMN24007732 lck eGFP Thymus 1 .filtered feature bc matrix.h5 GSM5732078 Adult Thymus 2 SRX13397706 SAMN24007733 lck eGFP Thymus 2 .filtered feature bc matrix.h5 GSM5732079 Juvenile Thymus 1 SRX13397707 SAMN24007734 4wpfThymus 1.filtered feature bc matrix.h5 GSM5732080 Juvenile Thymus 2 SRX13397708 SAMN24007735 4wpfThymus 3.filtered feature bc matrix.h5 GSM5732081 Juvenile Thymus 3 SRX13397709 SAMN24007736 4wpfThymus 5.filtered feature bc matrix.h5 GSM5732082 Juvenile Thymus 4 SRX13397710 SAMN24007737 4wpfThymus 6.filtered feature bc matrix.h5", null, null, null, "adult thy untreated", "GSM8115869", null, "tissue:13 wpf wpf loc name:missing|collection date:missing", "adult thy untreated", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 7.0.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Assembly: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: count matrices from the NovaSeq platforms", "13 wpf", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "tissue:13 wpf", "GSM8115869", "GSM8115869: adult thy untreated; Danio rerio; RNA Seq", "GSM8115869 r1", "GSM8115869", "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP492124", null, "loader:fastq load.py", "adult_thy_untreated_S5_L001_R2_001.fastq.gz adult_thy_untreated_S5_L001_R1_001.fastq.gz adult_thy_untreated_S5_L001_I1_001.fastq.gz", "fastq fastq fastq", 61867658967.0, 487146921.0, "GSM8115869 r1", "0:8 1:28 2:91", "A:12933400034;C:9373762639;G:10188374364;T:11834295354;N:537420", 8, 28, 91, null, 12933400034, 9373762639, 10188374364, 11834295354, 537420, "SRX23770350", "SRS20595835", null, null, "Oncology/Hematology, Boston Children's Hospital", 1, 0.90847, null, 0.19603, null, 0.76895, null, 0.56494, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-02-27", "Multi-stage", "Multi-stage", "Undetermined", "Undetermined"], [30663, "SRR28125660", "SRX23770349", "SRS20595836", "SRP492124", "PRJNA1081449", "Thymic mimetic cells in zebrafish", "GSE259418", "Transcriptome Analysis", "We profiled the zebrafish thymus by single cell RNA sequencing to investigate the heterogeneity of zebrafish thymic epithelial cells and determine whether similar mimetic cell populations to those found in mice and humans might also exist in the zebrafish. Overall design: To assess the heterogeneity of zebrafish thymic epithelial cells a total of 11 zebrafish samples derived from 14 adult and 21 juvenile zebrafish were studied. Specifically we re processed single cell RNA sequencing scRNA seq data from 8 zebrafish thymus samples derived from 4 adult zebrafish and 21 juvenile zebrafish previously described in GSE190794 GSM5732067 GSM5732070 GSM5732077 GSM5732078 GSM5732079 GSM5732080 GSM5732081 GSM5732082 so that reads mapping to intronic regions were included in the analysis to increase sensitivity and be consistent with mouse and human scRNA seq studies for cross species comparison. In addition we generated 3 new scRNA seq datasets derived from a total of 10 adult lck:mCherry;cd79a:eGFP transgenic zebrafish. The thymi from 5 of these adult zebrafish were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII whereas thymi from the other 5 adult zebrafish underwent enzymatic dissociation with Liberase TM prior to quenching filtering and cell sorting. The live non autofluorescent GFP negative mCherry negative cells a population depleted of T and B cells thus providing a relative enrichment of thymic epithelial cells were sorted and collected for 10x scRNA seq. One 10x lane was used for the mechanically dissociated sample untreated and two lanes were used for the liberase treated sample to obtain 2 technical replicates. Re analysed Samples in GSE190794/PRJNA788489 and their processed data file: SampleName Experiment BioSample processed data file GSM5732067 Adult Thymus 3 SRX13397695 SAMN24007722 Fish 22 Thymus .filtered feature bc matrix.h5 GSM5732070 Adult Thymus 4 SRX13397698 SAMN24007725 Fish 24 Thymus .filtered feature bc matrix.h5 GSM5732077 Adult Thymus 1 SRX13397705 SAMN24007732 lck eGFP Thymus 1 .filtered feature bc matrix.h5 GSM5732078 Adult Thymus 2 SRX13397706 SAMN24007733 lck eGFP Thymus 2 .filtered feature bc matrix.h5 GSM5732079 Juvenile Thymus 1 SRX13397707 SAMN24007734 4wpfThymus 1.filtered feature bc matrix.h5 GSM5732080 Juvenile Thymus 2 SRX13397708 SAMN24007735 4wpfThymus 3.filtered feature bc matrix.h5 GSM5732081 Juvenile Thymus 3 SRX13397709 SAMN24007736 4wpfThymus 5.filtered feature bc matrix.h5 GSM5732082 Juvenile Thymus 4 SRX13397710 SAMN24007737 4wpfThymus 6.filtered feature bc matrix.h5", null, null, null, "adult thy liberase 2", "GSM8115868", null, "tissue:13 wpf wpf loc name:missing|collection date:missing", "adult thy liberase 2", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 7.0.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Assembly: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: count matrices from the NovaSeq platforms", "13 wpf", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "tissue:13 wpf", "GSM8115868", "GSM8115868: adult thy liberase 2; Danio rerio; RNA Seq", "GSM8115868 r1", "GSM8115868", "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP492124", null, "loader:fastq load.py", "adult_thy_liberase_2_S7_L001_I1_001.fastq.gz adult_thy_liberase_2_S7_L001_R1_001.fastq.gz adult_thy_liberase_2_S7_L001_R2_001.fastq.gz", "fastq fastq fastq", 64816880653.0, 510369139.0, "GSM8115868 r1", "0:8 1:28 2:91", "A:12985717325;C:10400471721;G:10926930853;T:12129907262;N:564488", 8, 28, 91, null, 12985717325, 10400471721, 10926930853, 12129907262, 564488, "SRX23770349", "SRS20595836", null, null, "Oncology/Hematology, Boston Children's Hospital", 1, 0.90014, null, 0.13619, null, 0.81103, null, 0.54906, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-02-27", "Multi-stage", "Multi-stage", "Undetermined", "Undetermined"], [30664, "SRR28125661", "SRX23770348", "SRS20595834", "SRP492124", "PRJNA1081449", "Thymic mimetic cells in zebrafish", "GSE259418", "Transcriptome Analysis", "We profiled the zebrafish thymus by single cell RNA sequencing to investigate the heterogeneity of zebrafish thymic epithelial cells and determine whether similar mimetic cell populations to those found in mice and humans might also exist in the zebrafish. Overall design: To assess the heterogeneity of zebrafish thymic epithelial cells a total of 11 zebrafish samples derived from 14 adult and 21 juvenile zebrafish were studied. Specifically we re processed single cell RNA sequencing scRNA seq data from 8 zebrafish thymus samples derived from 4 adult zebrafish and 21 juvenile zebrafish previously described in GSE190794 GSM5732067 GSM5732070 GSM5732077 GSM5732078 GSM5732079 GSM5732080 GSM5732081 GSM5732082 so that reads mapping to intronic regions were included in the analysis to increase sensitivity and be consistent with mouse and human scRNA seq studies for cross species comparison. In addition we generated 3 new scRNA seq datasets derived from a total of 10 adult lck:mCherry;cd79a:eGFP transgenic zebrafish. The thymi from 5 of these adult zebrafish were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII whereas thymi from the other 5 adult zebrafish underwent enzymatic dissociation with Liberase TM prior to quenching filtering and cell sorting. The live non autofluorescent GFP negative mCherry negative cells a population depleted of T and B cells thus providing a relative enrichment of thymic epithelial cells were sorted and collected for 10x scRNA seq. One 10x lane was used for the mechanically dissociated sample untreated and two lanes were used for the liberase treated sample to obtain 2 technical replicates. Re analysed Samples in GSE190794/PRJNA788489 and their processed data file: SampleName Experiment BioSample processed data file GSM5732067 Adult Thymus 3 SRX13397695 SAMN24007722 Fish 22 Thymus .filtered feature bc matrix.h5 GSM5732070 Adult Thymus 4 SRX13397698 SAMN24007725 Fish 24 Thymus .filtered feature bc matrix.h5 GSM5732077 Adult Thymus 1 SRX13397705 SAMN24007732 lck eGFP Thymus 1 .filtered feature bc matrix.h5 GSM5732078 Adult Thymus 2 SRX13397706 SAMN24007733 lck eGFP Thymus 2 .filtered feature bc matrix.h5 GSM5732079 Juvenile Thymus 1 SRX13397707 SAMN24007734 4wpfThymus 1.filtered feature bc matrix.h5 GSM5732080 Juvenile Thymus 2 SRX13397708 SAMN24007735 4wpfThymus 3.filtered feature bc matrix.h5 GSM5732081 Juvenile Thymus 3 SRX13397709 SAMN24007736 4wpfThymus 5.filtered feature bc matrix.h5 GSM5732082 Juvenile Thymus 4 SRX13397710 SAMN24007737 4wpfThymus 6.filtered feature bc matrix.h5", null, null, null, "adult thy liberase 1", "GSM8115867", null, "tissue:13 wpf wpf loc name:missing|collection date:missing", "adult thy liberase 1", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 7.0.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Assembly: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: count matrices from the NovaSeq platforms", "13 wpf", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "tissue:13 wpf", "GSM8115867", "GSM8115867: adult thy liberase 1; Danio rerio; RNA Seq", "GSM8115867 r1", "GSM8115867", "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi into 0.5% BSA in HBSS without xxx or magnesium at room temperature. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Sorted cells were assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended in 0.5% BSA in DPBS. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of between 4 000 and 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP492124", null, "loader:fastq load.py", "adult_thy_liberase_1_S6_L001_I1_001.fastq.gz adult_thy_liberase_1_S6_L001_R1_001.fastq.gz adult_thy_liberase_1_S6_L001_R2_001.fastq.gz", "fastq fastq fastq", 62862684917.0, 494981771.0, "GSM8115867 r1", "0:8 1:28 2:91", "A:12631971877;C:10077821577;G:10579831603;T:11753169367;N:546737", 8, 28, 91, null, 12631971877, 10077821577, 10579831603, 11753169367, 546737, "SRX23770348", "SRS20595834", null, null, "Oncology/Hematology, Boston Children's Hospital", 1, 0.89921, null, 0.13602, null, 0.814, null, 0.55224, null, 91, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2024-02-27", "Multi-stage", "Multi-stage", "Undetermined", "Undetermined"], [33008, "SRR29632270", "SRX25138332", "SRS21830414", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 24h", "GSM8366755", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 24h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366755", "GSM8366755: Bmp4 explant 24h; Danio rerio; RNA Seq", "GSM8366755 r1", "GSM8366755", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4_24h_S1_L001_R1_001.fastq.gz Bmp4_24h_S1_L001_R2_001.fastq.gz", "fastq fastq", 49098784294.0, 412594826.0, "GSM8366755 r1", "0:28 1:91", "A:13643133254;C:11126085942;G:11676909916;T:12651522663;N:1132519", 28, 91, null, null, 13643133254, 11126085942, 11676909916, 12651522663, 1132519, "SRX25138332", "SRS21830414", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33009, "SRR29632271", "SRX25138331", "SRS21830413", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 18h", "GSM8366754", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 18h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366754", "GSM8366754: Bmp4 explant 18h; Danio rerio; RNA Seq", "GSM8366754 r1", "GSM8366754", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4_18h_S1_L001_R1_001.fastq.gz Bmp4_18h_S1_L001_R2_001.fastq.gz", "fastq fastq", 44863080801.0, 377000679.0, "GSM8366754 r1", "0:28 1:91", "A:12441486571;C:10199111519;G:10790306255;T:11430847129;N:1329327", 28, 91, null, null, 12441486571, 10199111519, 10790306255, 11430847129, 1329327, "SRX25138331", "SRS21830413", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33010, "SRR29632272", "SRX25138330", "SRS21830412", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 12h", "GSM8366753", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 12h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366753", "GSM8366753: Bmp4 explant 12h; Danio rerio; RNA Seq", "GSM8366753 r1", "GSM8366753", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4_12h_S1_L001_R1_001.fastq.gz Bmp4_12h_S1_L001_R2_001.fastq.gz", "fastq fastq", 50138128747.0, 421328813.0, "GSM8366753 r1", "0:28 1:91", "A:14089229753;C:10936347905;G:11403388741;T:13708020723;N:1141625", 28, 91, null, null, 14089229753, 10936347905, 11403388741, 13708020723, 1141625, "SRX25138330", "SRS21830412", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33011, "SRR29632273", "SRX25138329", "SRS21830410", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 10h", "GSM8366752", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 10h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366752", "GSM8366752: Bmp4 explant 10h; Danio rerio; RNA Seq", "GSM8366752 r1", "GSM8366752", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4-10h_S1_L001_R1_001.fastq.gz Bmp4-10h_S1_L001_R2_001.fastq.gz", "fastq fastq", 149164547700.0, 497215159.0, "GSM8366752 r1", "0:150 1:150", "A:44020073879;C:31344780625;G:29505876494;T:44289819189;N:3997513", 150, 150, null, null, 44020073879, 31344780625, 29505876494, 44289819189, 3997513, "SRX25138329", "SRS21830410", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33012, "SRR29632274", "SRX25138328", "SRS21830409", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 8h", "GSM8366751", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 8h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366751", "GSM8366751: Bmp4 explant 8h; Danio rerio; RNA Seq", "GSM8366751 r1", "GSM8366751", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4-8h_S1_L001_R1_001.fastq.gz Bmp4-8h_S1_L001_R2_001.fastq.gz", "fastq fastq", 118709118600.0, 395697062.0, "GSM8366751 r1", "0:150 1:150", "A:35474636770;C:23305712896;G:22894585568;T:37031410017;N:2773349", 150, 150, null, null, 35474636770, 23305712896, 22894585568, 37031410017, 2773349, "SRX25138328", "SRS21830409", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "B", "B", "mate1-mate2 similar by mapping diff", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33013, "SRR29632275", "SRX25138327", "SRS21830411", "SRP516621", "PRJNA1129168", "in vitro construction of an embryonic caudal organizer", "GSE270989", "Other", "Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf 8 hpf 10 hpf 12 hpf 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell three primeLibrary & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery. Results: paraxial mesoderm lateral plate mesoderm and their derivatives were detected in bmp4 explants and NMP somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well organized caudal like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids. Overall design: bmp4 injected zebrafish explants or BMP4 induced human embryoids were harvested for scRNA seq.", null, null, null, "Bmp4 explant 6h", "GSM8366750", null, "source name:embryonic|tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell|geo loc name:missing|collection date:missing", "Bmp4 explant 6h", "Illumina sequencing reads were aligned to the zebrafish GRCz11 or human mRNA reference genome GRCh38 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 GRCh38 Supplementary files format and content: tar archive includes filtered gene bc matrices post running CellRanger pipeline", "embryonic", "To generate bmp4 explants bmp4 8 10 pg mRNA was injected into one animal pole blastomere at the 128 cell stage. Then the animal pole region was explanted and raised to 24hpf. To generate BMP4 induced human embryoids human embryonic stem cells hESCs of the H9 line were grown on Matrigel coated 12 well plates for a period ranging from 48 to 72 hours utilizing them when they reached an approximate confluence of 70%. On the initial day of the experiment both wild type H9 cells and ihBmp4 cells were treated with accutase to facilitate their dissociation. They were then transferred to 96 well U bottom low attachment plates for culture. The seeding density was adjusted to 1300 cells per well for H9 cells and 400 cells per well for ihBmp4 cells using 50 \u03bcl of an aggregation medium based on TeSR E6 and supplemented with the ROCK inhibitor 10\u03bcM Y27632. post a 24 hour incubation period both types of cells formed embryoid bodies EBs in their respective wells. These H9 and ihBmp4 EBs were then co cultured in the same well with 80 \u03bcl of the TeSR E6 based medium to encourage fusion. Between days 2 and 4 of the culture period the EBs were subjected to an induction process to enhance Bmp4 expression which initiated the morphogenesis phase. The culture medium was replaced with a fresh TeSR E6 medium that included 75 ng ml 1 Doxycycline and 50 ng ml 1 FGF2 to stimulate these processes. Starting from day 4 the Doxycycline and FGF2 were omitted from the culture medium. The EBs were then maintained in a TeSR E6 based medium for continued growth and development.", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer\u2019s protocol for 10000 cell recovery.", "Bmp4 explants were cultured in Dulbecco's modified Eagle\u2019s medium DMEM/F 12 with 10 mM HEPES 1x MEM with non essential amino acids 7 mM CaCl2 1 mM sodium pyruvate 50 \u03bcg/ml gentamycin 100 \u03bcM 2 mercaptoethanol 1\u00d7 antibiotic antimycotic 10% serum replacement and kept at 28.5\u2103.BMP4 induced human embryoids were cultured in a 96 well U shaped ultra low plate with culture medium TeSR E6 medium supplemented with 0.5% N2 and 0.5% B27 to support cell growth. Small molecule compounds and recombinant proteins inducing differentiation were added according to the treatment protocol. Samples were collected on day 5 and 6 of differentiation.", "tissue:embryonic|cell line:zebrafish embryonic stem cells|cell type:stem cell", "GSM8366750", "GSM8366750: Bmp4 explant 6h; Danio rerio; RNA Seq", "GSM8366750 r1", "GSM8366750", "1", "Single cell RNA of each sample was extracted by following the Chromium Controller and Chromium Single Cell 30 Library & Gel Bead Kit v3.110x Genomics PN 1000121 protocol. Libraries were prepared using Single Cell 3\u2032Library & gel Bead kit v3.1 10x Genomics Cat# PN 1000121 according to the manufacturer's protocol for 10000 cell recovery.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP516621", null, null, "Bmp4_6h_S1_L001_R1_001.fastq.gz Bmp4_6h_S1_L001_R2_001.fastq.gz", "fastq fastq", 52586645972.0, 441904588.0, "GSM8366750 r1", "0:28 1:91", "A:14755432217;C:11271293276;G:11858476334;T:14700289448;N:1154697", 28, 91, null, null, 14755432217, 11271293276, 11858476334, 14700289448, 1154697, "SRX25138327", "SRS21830411", "SRA1911917", "Institute of genetics, Zhejiang University", "Institute of genetics, Zhejiang University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-06-27", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"], [33669, "SRR30530658", "SRX25954305", "SRS22536191", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0chigh glucose", "GSM8494124", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0chigh glucose", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494124", "GSM8494124: endothelial cells\uff0chigh glucose; Danio rerio; RNA Seq", "GSM8494124 r1", "GSM8494124", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "lr57glu96hpf_R1.fq.gz lr57glu96hpf_R2.fq.gz", "fastq fastq", 196891371600.0, 656304572.0, "GSM8494124 r1", "0:150 1:150", "A:46034913013;C:37262343828;G:60636104680;T:52955473756;N:2536323", 150, 150, null, null, 46034913013, 37262343828, 60636104680, 52955473756, 2536323, "SRX25954305", "SRS22536191", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33670, "SRR30530659", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_1_1_R1.fq.gz SH190424C_LR5796dpf_1_1_R2.fq.gz", "fastq fastq", 18351420300.0, 61171401.0, "GSM8494123 r1", "0:150 1:150", "A:3872504772;C:3434009784;G:6583451920;T:4461384217;N:69607", 150, 150, null, null, 3872504772, 3434009784, 6583451920, 4461384217, 69607, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33671, "SRR30530660", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_1_2_R1.fq.gz SH190424C_LR5796dpf_1_2_R2.fq.gz", "fastq fastq", 36386960400.0, 121289868.0, "GSM8494123 r2", "0:150 1:150", "A:8890006445;C:6974694151;G:10814990376;T:9707137150;N:132278", 150, 150, null, null, 8890006445, 6974694151, 10814990376, 9707137150, 132278, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33672, "SRR30530661", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_2_1_R1.fq.gz SH190424C_LR5796dpf_2_1_R2.fq.gz", "fastq fastq", 44811813000.0, 149372710.0, "GSM8494123 r3", "0:150 1:150", "A:10939691275;C:8613307673;G:13332283843;T:11926366839;N:163370", 150, 150, null, null, 10939691275, 8613307673, 13332283843, 11926366839, 163370, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33673, "SRR30530662", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_2_2_R1.fq.gz SH190424C_LR5796dpf_2_2_R2.fq.gz", "fastq fastq", 22587582300.0, 75291941.0, "GSM8494123 r4", "0:150 1:150", "A:4762937825;C:4235264474;G:8108549401;T:5480746863;N:83737", 150, 150, null, null, 4762937825, 4235264474, 8108549401, 5480746863, 83737, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33674, "SRR30530663", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_3_1_R1.fq.gz SH190424C_LR5796dpf_3_1_R2.fq.gz", "fastq fastq", 31458502200.0, 104861674.0, "GSM8494123 r5", "0:150 1:150", "A:7597642424;C:6175910515;G:9377029875;T:8307805635;N:113751", 150, 150, null, null, 7597642424, 6175910515, 9377029875, 8307805635, 113751, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33675, "SRR30530664", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_3_2_R1.fq.gz SH190424C_LR5796dpf_3_2_R2.fq.gz", "fastq fastq", 15812987400.0, 52709958.0, "GSM8494123 r6", "0:150 1:150", "A:3317721700;C:3009997461;G:5662825685;T:3822382737;N:59817", 150, 150, null, null, 3317721700, 3009997461, 5662825685, 3822382737, 59817, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", "Multi-stage", "Endothelium", "Cardiovascular System"], [33676, "SRR30530665", "SRX25954304", "SRS22536190", "SRP530279", "PRJNA1156090", "Single cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated zebrafish embryos", "GSE276251", "Transcriptome Analysis", "Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar sweetened beverages. Nevertheless the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. Presently we have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose. Utilizing this model we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels especially intersegmental vessels ISVs. The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells ECs into tip cells. Single cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs proliferating ECs and a series of upregulated proangiogenic genes. Overall design: Firstly we isolated the EGFP positive cells from control and high glucose treated Tgfli1ep:EGFP CAAX transgenic embryos. Following the proteolytic dissociation of embryos the EGFP positive cells were isolated by fluorescence activated cell sorting FACS. Around 300500 zebrafish embryos were used for the ECs collection for each stage. The isolated ECs were analyzed using a large scale scRNA seq 10X Genomics platform.", null, "pubmed:39365738;pubmed:39882604", null, "endothelial cells\uff0ccontrol", "GSM8494123", null, "tissue:endothelial cells|cell type:endothelial cells|geo loc name:missing|collection date:missing", "endothelial cells\uff0ccontrol", "Cell Ranger 3.0.2 https://github.com/10XGenomics/cellranger was used to convert the raw sequencing data to single cell level gene counts matrix. The clustering of single cells and the marker genes in each cluster were analyzed by Seurat 3.0 https://satijalab.org/seurat/install.html Assembly: The reference genome fasta format and gene annotation gtf format files were downloaded from Ensembl database release version 94. Supplementary files format and content: Tab separated values files and matrix files.", "endothelial cells", "Embryos were treated with 6% glucose from 24 hpf to 72 hpf.", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell 3\u2019Reagent Kits v3 from 10X Genomics Company.", "Tgkdrl:EGFP embryos were raised in E3 solution at 28.5\u2103 to the indicated developmental stage.", "cell type:endothelial cells", "GSM8494123", "GSM8494123: endothelial cells\uff0ccontrol; Danio rerio; RNA Seq", "GSM8494123 r1", "GSM8494123", "1", "Embryos were transferred into a 15 mL falcon tube with 10 mL phosphate buffered saline PBS containing 0.25% trypsin and incubated in a rolling machine for 30 min at 28\u00b0C during which they were pipetted up and down per 5 min. post centrifuging for 5 min at 800 g at 4\u00b0C cells were resuspended in PBS containing 0.25% trypsin and 15% fetal calf serum FCS to stop the digestion and centrifuged for 5 min at 800 g at 4\u00b0C. Cells were rinsed with PBS containing 2% FCS for 2 times and resuspended in PBS. EGFP positive cells were first enriched by fluorescence activated cell sorting FACS and then went through library preparation following the standard protocol named Chromium Single Cell three primeReagent Kits v3 from 10X Genomics Company.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 4000", null, "SRP530279", null, null, "SH190424C_LR5796dpf_4_1_R1.fq.gz SH190424C_LR5796dpf_4_1_R2.fq.gz", "fastq fastq", 19472976900.0, 64909923.0, "GSM8494123 r7", "0:150 1:150", "A:4109297307;C:3636046624;G:6996008921;T:4731548865;N:75183", 150, 150, null, null, 4109297307, 3636046624, 6996008921, 4731548865, 75183, "SRX25954304", "SRS22536190", "SRA1961613", "Nantong University", "Nantong University", null, null, null, null, null, null, null, null, null, null, null, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "China", "2024-09-03", "Multi-stage", 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