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This submission includes reads tagged with the sequence CGATGT.", "RNA seq dUTP eukaryotic", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP016002", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16", "20417_1#2.cram", "cram", 581391900.0, 3875946.0, "SC RUN 20417 1#2", "0:75 1:75", "A:156042125;C:134966129;G:135462829;T:154862718;N:58099", 75, 75, null, null, 156042125, 134966129, 135462829, 154862718, 58099, "ERX1776742", "ERS1226745", "ERA740537", "European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.9582, 0.96234, 0.1297, 0.12747, 0.72863, 0.72949, 0.48838, 0.48132, 75, 75, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2016-06-28", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [5048, "ERR1706507", "ERX1776741", "ERS1226722", "ERP016002", "PRJEB14364", "Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos", "Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266", "Transcriptome Analysis", "RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.", "ArrayExpress:E ERAD 507", null, null, "zmp ph260 A1", "SAMEA4055612", "Wellcome Sanger Institute", "ArrayExpress DevelopmentalStage:Segmentation:26+somites   Pharyngula:Prim 5   ZFS:0000028   ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055612|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:19:38Z|INSDC status:public|Submitter Id:d6e3f3e0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d6e3f3e0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 20417 1#1", "17117057", "Illumina sequencing of library 17117057  constructed from sample accession ERS1226722 for study accession ERP016002.  This is part of an Illumina multiplexed sequencing run 20417 1.  This submission includes reads tagged with the sequence ATCACG.", "RNA seq dUTP eukaryotic", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP016002", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16", "20417_1#1.cram", "cram", 1233271650.0, 8221811.0, "SC RUN 20417 1#1", "0:75 1:75", "A:334336992;C:282740616;G:283953463;T:332113804;N:126775", 75, 75, null, null, 334336992, 282740616, 283953463, 332113804, 126775, "ERX1776741", "ERS1226722", "ERA740537", "European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.95579, 0.95701, 0.15941, 0.15689, 0.71758, 0.71709, 0.4967, 0.5163, 75, 75, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2016-06-28", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [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. Libraries were constructed using the 10x v2 chemistry.", "Sample 2", "SAMEA6873729", "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:SAMEA6873729|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 2|age:8|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 2|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 2 s", "Sample 2 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. 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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:8|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", "cDNA8h_1_S2_L001.bam", "bam", 10087287534.0, 99874134.0, "E MTAB 9193:cDNA8h 1 S2 L001", "0:101", "A:2892833391;C:2022466595;G:2198646699;T:2962268446;N:11072403", 101, null, null, null, 2892833391, 2022466595, 2198646699, 2962268446, 11072403, "ERX4155254", "ERS4601292", "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, 0.93609, null, 0.12902, null, 0.82158, null, 0.5062, 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"], [9899, "ERR4194115", "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. Libraries were constructed using the 10x v2 chemistry.", "Sample 2", "SAMEA6873729", "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:SAMEA6873729|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 2|age:8|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 2|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 2 s", "Sample 2 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:8|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", "cDNA8h_1_S2_L002.bam", "bam", 10191911414.0, 100910014.0, "E MTAB 9193:cDNA8h 1 S2 L002", "0:101", "A:2923216190;C:2043975715;G:2221734566;T:2992798366;N:10186577", 101, null, null, null, 2923216190, 2043975715, 2221734566, 2992798366, 10186577, "ERX4155254", "ERS4601292", "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, 0.93445, null, 0.13038, null, 0.824, null, 0.49774, 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"], [9900, "ERR4194112", "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_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"], [10213, "ERR6511329", "ERX6138165", "ERS7264188", "ERP131213", "PRJEB46978", "Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore sequencing", "ena-STUDY-CENTER FOR GENOMIC REGULATION (CRG)-12-08-2021-14:48:52:906-1159", "Other", "Nano3P seq is a simple and robust method to accurately estimate transcript levels  tail lengths and tail nucleotide composition information in full length individual reads  with minimal library preparation biases  both in the coding and non coding transcriptome.", "ENA FIRST PUBLIC:2023 12 28|ENA LAST UPDATE:2023 12 28", null, "Zebrafish Nano3P seq of Ribodepleted sample biological replicate 1 including 2 hpf 4 hpf 6 hpf RNAs", "Zebrafish Ribodep Rep1", "SAMEA9541418", "CENTER FOR GENOMIC REGULATION (CRG)", "ENA FIRST PUBLIC:2023 12 28T01:07:23Z|ENA LAST UPDATE:2023 12 28T01:07:23Z|External Id:SAMEA9541418|INSDC center name:CENTER FOR GENOMIC REGULATION CRG|INSDC first public:2023 12 28T01:07:23Z|INSDC last update:2023 12 28T01:07:23Z|INSDC status:public|Submitter Id:Zebrafish Ribodep Rep1|common name:zebrafish|sample name:Zebrafish Ribodep Rep1|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "MinION sequencing", "ena EXPERIMENT CENTER FOR GENOMIC REGULATION CRG 17 08 2021 13:09:55:665 3", "cDNA786327", "Nano3P seq", "Nano3P seq", null, "OTHER", "TRANSCRIPTOMIC", "unspecified", "SINGLE", "OXFORD_NANOPORE", "MinION", null, "ERP131213", "MinION sequencing", "ENA FIRST PUBLIC:2023 12 28|ENA LAST UPDATE:2023 12 28", "zebrafish_ribodep_rep1.tar.gz", "nanopore", 1745399583.0, 1644167.0, "ena RUN CENTER FOR GENOMIC REGULATION CRG 17 08 2021 13:09:55:665 3", "0:1061.57", "A:449704009;C:421915878;G:378879857;T:494899839;N:0", 1061, null, null, null, 449704009, 421915878, 378879857, 494899839, 0, "ERX6138165", "ERS7264188", "ERA5757997", "CENTER FOR GENOMIC REGULATION (CRG)|European Nucleotide Archive", "CENTER FOR GENOMIC REGULATION (CRG)", 1, 0.01112, null, 0.0, null, 0.99997, null, 1.0, null, 546, null, "T", null, "long read", "ont", "ont", "full_length", "rrna_depletion", "unknown", "bulk", "unknown", "unknown", null, "Spain", "2023-12-28", "Multi-stage", "Embryo", "Undetermined", "Embryo Imprecise"], [10215, "ERR6511330", "ERX6138166", "ERS7264189", "ERP131213", "PRJEB46978", "Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore sequencing", "ena-STUDY-CENTER FOR GENOMIC REGULATION (CRG)-12-08-2021-14:48:52:906-1159", "Other", "Nano3P seq is a simple and robust method to accurately estimate transcript levels  tail lengths and tail nucleotide composition information in full length individual reads  with minimal library preparation biases  both in the coding and non coding transcriptome.", "ENA FIRST PUBLIC:2023 12 28|ENA LAST UPDATE:2023 12 28", null, "Zebrafish Nano3P seq of Ribodepleted sample biological replicate 1 including 2 hpf 4 hpf 6 hpf RNAs", "Zebrafish Ribodep Rep2", "SAMEA9541419", "CENTER FOR GENOMIC REGULATION (CRG)", "ENA FIRST PUBLIC:2023 12 28T01:07:23Z|ENA LAST UPDATE:2023 12 28T01:07:23Z|External Id:SAMEA9541419|INSDC center name:CENTER FOR GENOMIC REGULATION CRG|INSDC first public:2023 12 28T01:07:23Z|INSDC last update:2023 12 28T01:07:23Z|INSDC status:public|Submitter Id:Zebrafish Ribodep Rep2|common name:zebrafish|sample name:Zebrafish Ribodep Rep2|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "MinION sequencing", "ena EXPERIMENT CENTER FOR GENOMIC REGULATION CRG 17 08 2021 13:09:55:665 4", "cDNA123791", "Nano3P seq", "Nano3P seq", null, "OTHER", "TRANSCRIPTOMIC", "unspecified", "SINGLE", "OXFORD_NANOPORE", "MinION", null, "ERP131213", "MinION sequencing", "ENA FIRST PUBLIC:2023 12 28|ENA LAST UPDATE:2023 12 28", "zebrafish_ribodep_rep2.tar.gz", "nanopore", 2038398139.0, 1955617.0, "ena RUN CENTER FOR GENOMIC REGULATION CRG 17 08 2021 13:09:55:665 4", "0:1042.33", "A:518369802;C:477535545;G:441294056;T:601198736;N:0", 1042, null, null, null, 518369802, 477535545, 441294056, 601198736, 0, "ERX6138166", "ERS7264189", "ERA5757997", "CENTER FOR GENOMIC REGULATION (CRG)|European Nucleotide Archive", "CENTER FOR GENOMIC REGULATION (CRG)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, "ont", "ont", "full_length", "rrna_depletion", "unknown", "bulk", "unknown", "unknown", null, "Spain", "2023-12-28", "Multi-stage", "Embryo", "Undetermined", "Embryo Imprecise"], [10649, "ERR406885", "ERX373262", "ERS391761", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5s24", "SAMEA2299303", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299303|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:5s24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5s24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.4", "batchA 24hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5s24.fq.gz", "fastq", 584550600.0, 11691012.0, "E MTAB 2194:5s24.fq.gz", "0:50 1:0", "A:154602150;C:138134763;G:138628704;T:153175234;N:9749", 50, 0, null, null, 154602150, 138134763, 138628704, 153175234, 9749, "ERX373262", "ERS391761", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.9356, null, 0.0996, null, 0.68615, null, 0.47069, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10650, "ERR406893", "ERX373261", "ERS391760", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5y32", "SAMEA2299302", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299302|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:5y32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5y32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.5", "batchA 32hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5y32.fq.gz", "fastq", 528892950.0, 10577859.0, "E MTAB 2194:5y32.fq.gz", "0:50 1:0", "A:140383478;C:125036983;G:124119152;T:139344643;N:8694", 50, 0, null, null, 140383478, 125036983, 124119152, 139344643, 8694, "ERX373261", "ERS391760", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93478, null, 0.10542, null, 0.68296, null, 0.4795, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10651, "ERR406898", "ERX373260", "ERS391759", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6s24", "SAMEA2299301", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299301|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:6s24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6s24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.10", "batchB 24hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6s24.fq.gz", "fastq", 542771850.0, 10855437.0, "E MTAB 2194:6s24.fq.gz", "0:50 1:0", "A:142311943;C:129686202;G:129130172;T:141634727;N:8806", 50, 0, null, null, 142311943, 129686202, 129130172, 141634727, 8806, "ERX373260", "ERS391759", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93717, null, 0.09239, null, 0.68876, null, 0.46809, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10652, "ERR406896", "ERX373259", "ERS391758", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13y32", "SAMEA2299300", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299300|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:13y32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13y32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.18", "batchC 32hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13y32.fq.gz", "fastq", 649898350.0, 12997967.0, "E MTAB 2194:13y32.fq.gz", "0:50 1:0", "A:169738689;C:156130165;G:155102788;T:168916174;N:10534", 50, 0, null, null, 169738689, 156130165, 155102788, 168916174, 10534, "ERX373259", "ERS391758", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93898, null, 0.08535, null, 0.68217, null, 0.47133, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10653, "ERR406899", "ERX373258", "ERS391757", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5s32", "SAMEA2299299", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299299|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:5s32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5s32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.6", "batchA 32hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5s32.fq.gz", "fastq", 566813600.0, 11336272.0, "E MTAB 2194:5s32.fq.gz", "0:50 1:0", "A:149907366;C:134421172;G:133399280;T:149076590;N:9192", 50, 0, null, null, 149907366, 134421172, 133399280, 149076590, 9192, "ERX373258", "ERS391757", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93497, null, 0.10019, null, 0.68379, null, 0.4671, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10654, "ERR406888", "ERX373257", "ERS391756", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5y24", "SAMEA2299298", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299298|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:5y24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5y24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.3", "batchA 24hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5y24.fq.gz", "fastq", 578936250.0, 11578725.0, "E MTAB 2194:5y24.fq.gz", "0:50 1:0", "A:154884020;C:135497257;G:135214353;T:153331273;N:9347", 50, 0, null, null, 154884020, 135497257, 135214353, 153331273, 9347, "ERX373257", "ERS391756", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.9342, null, 0.1106, null, 0.67811, null, 0.47685, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10655, "ERR406887", "ERX373256", "ERS391755", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13s24", "SAMEA2299297", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299297|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:13s24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13s24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.16", "batchC 24hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13s24.fq.gz", "fastq", 837415050.0, 16748301.0, "E MTAB 2194:13s24.fq.gz", "0:50 1:0", "A:217172462;C:202556518;G:201365353;T:216307386;N:13331", 50, 0, null, null, 217172462, 202556518, 201365353, 216307386, 13331, "ERX373256", "ERS391755", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93888, null, 0.07561, null, 0.6899, null, 0.46646, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10656, "ERR406894", "ERX373255", "ERS391754", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6y24", "SAMEA2299296", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299296|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:6y24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6y24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.9", "batchB 24hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6y24.fq.gz", "fastq", 569424550.0, 11388491.0, "E MTAB 2194:6y24.fq.gz", "0:50 1:0", "A:149287318;C:136121879;G:135445765;T:148560272;N:9316", 50, 0, null, null, 149287318, 136121879, 135445765, 148560272, 9316, "ERX373255", "ERS391754", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93486, null, 0.0893, null, 0.68751, null, 0.46133, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10657, "ERR406895", "ERX373254", "ERS391753", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13s8", "SAMEA2299295", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299295|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:13s8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13s8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.14", "batchC 8hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13s8.fq.gz", "fastq", 532141500.0, 10642830.0, "E MTAB 2194:13s8.fq.gz", "0:50 1:0", "A:139773245;C:127051350;G:126773752;T:138534560;N:8593", 50, 0, null, null, 139773245, 127051350, 126773752, 138534560, 8593, "ERX373254", "ERS391753", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93506, null, 0.0742, null, 0.74748, null, 0.47765, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10658, "ERR406891", "ERX373253", "ERS391752", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6s8", "SAMEA2299294", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299294|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:6s8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6s8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.8", "batchB 8hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6s8.fq.gz", "fastq", 477620550.0, 9552411.0, "E MTAB 2194:6s8.fq.gz", "0:50 1:0", "A:127095393;C:112660046;G:111717464;T:126139686;N:7961", 50, 0, null, null, 127095393, 112660046, 111717464, 126139686, 7961, "ERX373253", "ERS391752", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93605, null, 0.08112, null, 0.74552, null, 0.46821, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10659, "ERR406886", "ERX373252", "ERS391751", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6y8", "SAMEA2299293", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:38Z|External Id:SAMEA2299293|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:38Z|INSDC status:public|Submitter Id:E MTAB 2194:6y8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6y8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.7", "batchB 8hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6y8.fq.gz", "fastq", 508637450.0, 10172749.0, "E MTAB 2194:6y8.fq.gz", "0:50 1:0", "A:135377293;C:119833844;G:119447157;T:133970958;N:8198", 50, 0, null, null, 135377293, 119833844, 119447157, 133970958, 8198, "ERX373252", "ERS391751", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93203, null, 0.07833, null, 0.73744, null, 0.47586, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10660, "ERR406892", "ERX373251", "ERS391750", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6y32", "SAMEA2299292", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:37Z|External Id:SAMEA2299292|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:37Z|INSDC status:public|Submitter Id:E MTAB 2194:6y32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6y32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.11", "batchB 32hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6y32.fq.gz", "fastq", 568482400.0, 11369648.0, "E MTAB 2194:6y32.fq.gz", "0:50 1:0", "A:149679881;C:135411801;G:134312216;T:149069222;N:9280", 50, 0, null, null, 149679881, 135411801, 134312216, 149069222, 9280, "ERX373251", "ERS391750", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93184, null, 0.104, null, 0.68128, null, 0.47652, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10661, "ERR406897", "ERX373250", "ERS391749", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5y8", "SAMEA2299291", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:37Z|External Id:SAMEA2299291|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:37Z|INSDC status:public|Submitter Id:E MTAB 2194:5y8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5y8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.1", "batchA 8hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5y8.fq.gz", "fastq", 497573200.0, 9951464.0, "E MTAB 2194:5y8.fq.gz", "0:50 1:0", "A:134128000;C:115688460;G:114668036;T:133080206;N:8498", 50, 0, null, null, 134128000, 115688460, 114668036, 133080206, 8498, "ERX373250", "ERS391749", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93519, null, 0.07057, null, 0.73511, null, 0.47977, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10662, "ERR406889", "ERX373249", "ERS391748", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13s32", "SAMEA2299290", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:37Z|External Id:SAMEA2299290|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:37Z|INSDC status:public|Submitter Id:E MTAB 2194:13s32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13s32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.19", "batchC 32hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13s32.fq.gz", "fastq", 637724150.0, 12754483.0, "E MTAB 2194:13s32.fq.gz", "0:50 1:0", "A:165500205;C:154383334;G:152953181;T:164877160;N:10270", 50, 0, null, null, 165500205, 154383334, 152953181, 164877160, 10270, "ERX373249", "ERS391748", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93998, null, 0.08954, null, 0.68146, null, 0.4729, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10663, "ERR406890", "ERX373248", "ERS391747", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13y24", "SAMEA2299289", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:26Z|External Id:SAMEA2299289|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:26Z|INSDC status:public|Submitter Id:E MTAB 2194:13y24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13y24|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.15", "batchC 24hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:24 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13y24.fq.gz", "fastq", 834725800.0, 16694516.0, "E MTAB 2194:13y24.fq.gz", "0:50 1:0", "A:216896952;C:201332171;G:200308143;T:216174965;N:13569", 50, 0, null, null, 216896952, 201332171, 200308143, 216174965, 13569, "ERX373248", "ERS391747", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.9358, null, 0.07569, null, 0.68757, null, 0.46994, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10664, "ERR406900", "ERX373247", "ERS391746", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "13y8", "SAMEA2299288", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:26Z|External Id:SAMEA2299288|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:26Z|INSDC status:public|Submitter Id:E MTAB 2194:13y8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:13y8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.13", "batchC 8hpf YD", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Removed yolk around 5hpf YD with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:yolk|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "13y8.fq.gz", "fastq", 457245150.0, 9144903.0, "E MTAB 2194:13y8.fq.gz", "0:50 1:0", "A:119648940;C:109589521;G:109491446;T:118507707;N:7536", 50, 0, null, null, 119648940, 109589521, 109491446, 118507707, 7536, "ERX373247", "ERS391746", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93146, null, 0.0681, null, 0.73718, null, 0.47464, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10665, "ERR406884", "ERX373246", "ERS391745", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "6s32", "SAMEA2299287", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:26Z|External Id:SAMEA2299287|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:26Z|INSDC status:public|Submitter Id:E MTAB 2194:6s32|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:6s32|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.12", "batchB 32hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:32 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "6s32.fq.gz", "fastq", 652353200.0, 13047064.0, "E MTAB 2194:6s32.fq.gz", "0:50 1:0", "A:170844587;C:156203331;G:155264927;T:170029850;N:10505", 50, 0, null, null, 170844587, 156203331, 155264927, 170029850, 10505, "ERX373246", "ERS391745", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93785, null, 0.09102, null, 0.68554, null, 0.46397, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [10666, "ERR406901", "ERX373245", "ERS391744", "ERP004564", "PRJEB5188", "YD embryos 8 hpf 24 hpf 32 hpf", "E-MTAB-2194", "Transcriptome Analysis", "Limited nutrient availability during development puts individuals at risk to develop complications later in life. Central in this early life stress paradox lies developmental plasticity  a poorly understood mechanism that responds to environmental cues from early to late developmental stages.  In this study  we introduce the zebrafish Danio rerio as a model to study the early developmental responses to reduced nutrient availability and their outcome. To reduce nutrient availability  we partially remove the yolk during embryogenesis. Around 5 hpf  we removed 30% of the yolk YD samples or sham punctured embryos SP with a Hamilton syringe system. At 8  24 hpf and 32 hpf  we collected RNA from whole embryos and obtained transcriptome profiles by RNAseq.", null, null, "Protocols: Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "5s8", "SAMEA2299286", "CAS-MPG PICB", "ENA FIRST PUBLIC:2014 04 01T17:00:44Z|ENA LAST UPDATE:2018 03 08T17:04:26Z|External Id:SAMEA2299286|INSDC center name:CAS MPG PICB|INSDC first public:2014 04 01T17:00:44Z|INSDC last update:2018 03 08T17:04:26Z|INSDC status:public|Submitter Id:E MTAB 2194:5s8|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo stage|genotype:wild type genotype|sample name:E MTAB 2194:5s8|scientific name:Danio rerio|specimen with known storage state:fresh specimen|strain:tb", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "E MTAB 2194:Unique sample ID.2", "batchA 8hpf SP", "YD embryos 8 hpf 24 hpf 32 hpf", "Zebrafish embryos in HANKS embryo buffer Around 5hpf  sham puncture the controls SP with hamilton syringe system. Trizol RNA extraction of whole embryos Truseq RNA Sample preparation Kits V2", "Experimental Factor: treatment:sham punctured embryos|Experimental Factor: time:8 hour", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP004564", "Illumina HiSeq 2000 sequencing; YD embryos 8 hpf 24 hpf 32 hpf", "ENA FIRST PUBLIC:2014 04 01|ENA LAST UPDATE:2018 11 16", "5s8.fq.gz", "fastq", 359298500.0, 7185970.0, "E MTAB 2194:5s8.fq.gz", "0:50 1:0", "A:96450828;C:83805020;G:83440885;T:95595844;N:5923", 50, 0, null, null, 96450828, 83805020, 83440885, 95595844, 5923, "ERX373245", "ERS391744", "ERA280282", "CAS-MPG PICB|ArrayExpress", "CAS-MPG PICB|ArrayExpress", 1, 0.93524, null, 0.07491, null, 0.73312, null, 0.47914, null, 50, null, "B", null, "usable mapping rate", "illumina", "hiseq_era", "unknown", "random_priming", "trueseq", "bulk", "unknown", "unknown", null, "China", "2014-04-01", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11042, "ERR9839781", "ERX9385638", "ERS12199238", "ERP138294", "PRJEB53494", "Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing", "94bf5509-4622-4d5f-b7c5-6a14bdfac340", "Other", "RNA polyadenylation plays a central role in RNA maturation  fate  and stability. In response to developmental cues  polyA tail lengths can vary  affecting the translation efficiency and stability of mRNAs. Here  we develop Nanopore three prime end capture sequencing Nano3P seq  a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance  tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol  Nano3P seq can sequence any given RNA molecule from its three prime end  regardless of its polyadenylation status  without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes  providing quantitative estimates of RNA abundance and tail lengths in mRNA  lncRNA  sn/snoRNA  scaRNA  and rRNA molecules. We find that  in addition to mRNA and lncRNA  polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover  we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level  correlating with mRNA decay. Finally  we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis.  Overall  Nano3P seq is a simple and robust method for accurately estimating transcript levels  tail lengths  and tail composition heterogeneity in individual reads  with minimal library preparation biases  both in the coding and non coding transcriptome.", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", null, "Zebrafish Ribodepleted RNA 2hpf  4hpf  6hpf", "Zebrafish Ribodepleted Rep3", "SAMEA110100413", "CENTER FOR GENOMIC REGULATION (CRG)", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10|External Id:SAMEA110100413|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG|INSDC center name:CENTER FOR GENOMIC REGULATION CRG|INSDC first public:2022 10 10T00:20:53Z|INSDC last update:2022 10 10T00:20:53Z|INSDC status:public|Submitter Id:Zebrafish Ribodepleted Rep3|common name:zebrafish|sample name:Zebrafish Ribodepleted Rep3", null, null, null, null, null, null, null, null, "MinION sequencing", "ena EXPERIMENT TAB 13 06 2022 16:07:52:807 817", "cDNA897892 ZFRDR3", "1", null, null, "RNA-Seq", "TRANSCRIPTOMIC", "other", "SINGLE", "OXFORD_NANOPORE", "MinION", null, "ERP138294", "MinION sequencing", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", "cDNA897892_ZFRDR3.tar.gz", "nanopore", 848659575.0, 587586.0, "ena RUN TAB 13 06 2022 16:07:52:808 818", "0:1444.32", "A:210205014;C:186527780;G:188214279;T:263712502;N:0", 1444, null, null, null, 210205014, 186527780, 188214279, 263712502, 0, "ERX9385638", "ERS12199238", "ERA15547404", "CENTER FOR GENOMIC REGULATION (CRG)|European Nucleotide Archive", "CENTER FOR GENOMIC REGULATION (CRG)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, "ont", "ont", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "Spain", "2022-10-10", "Multi-stage", "Embryo", "Undetermined", "Embryo Imprecise"], [11043, "ERR9839780", "ERX9385637", "ERS12199237", "ERP138294", "PRJEB53494", "Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing", "94bf5509-4622-4d5f-b7c5-6a14bdfac340", "Other", "RNA polyadenylation plays a central role in RNA maturation  fate  and stability. In response to developmental cues  polyA tail lengths can vary  affecting the translation efficiency and stability of mRNAs. Here  we develop Nanopore three prime end capture sequencing Nano3P seq  a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance  tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol  Nano3P seq can sequence any given RNA molecule from its three prime end  regardless of its polyadenylation status  without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes  providing quantitative estimates of RNA abundance and tail lengths in mRNA  lncRNA  sn/snoRNA  scaRNA  and rRNA molecules. We find that  in addition to mRNA and lncRNA  polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover  we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level  correlating with mRNA decay. Finally  we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis.  Overall  Nano3P seq is a simple and robust method for accurately estimating transcript levels  tail lengths  and tail composition heterogeneity in individual reads  with minimal library preparation biases  both in the coding and non coding transcriptome.", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", null, "Zebrafish Ribodepleted RNA 2hpf  4hpf  6hpf", "Zebrafish Ribodepleted Rep2", "SAMEA110100412", "CENTER FOR GENOMIC REGULATION (CRG)", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10|External Id:SAMEA110100412|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG|INSDC center name:CENTER FOR GENOMIC REGULATION CRG|INSDC first public:2022 10 10T00:20:53Z|INSDC last update:2022 10 10T00:20:53Z|INSDC status:public|Submitter Id:Zebrafish Ribodepleted Rep2|common name:zebrafish|sample name:Zebrafish Ribodepleted Rep2", null, null, null, null, null, null, null, null, "MinION sequencing", "ena EXPERIMENT TAB 13 06 2022 16:07:52:807 815", "cDNA123791 ZFRDR2", "1", null, null, "RNA-Seq", "TRANSCRIPTOMIC", "other", "SINGLE", "OXFORD_NANOPORE", "MinION", null, "ERP138294", "MinION sequencing", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", "cDNA123791_ZFRDR2.tar.gz", "nanopore", 2229275175.0, 1955617.0, "ena RUN TAB 13 06 2022 16:07:52:807 816", "0:1139.93", "A:533543922;C:498938137;G:515833119;T:680959997;N:0", 1139, null, null, null, 533543922, 498938137, 515833119, 680959997, 0, "ERX9385637", "ERS12199237", "ERA15547404", "CENTER FOR GENOMIC REGULATION (CRG)|European Nucleotide Archive", "CENTER FOR GENOMIC REGULATION (CRG)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, "ont", "ont", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "Spain", "2022-10-10", "Multi-stage", "Embryo", "Undetermined", "Embryo Imprecise"], [11044, "ERR9839779", "ERX9385636", "ERS12199236", "ERP138294", "PRJEB53494", "Nano3P seq: transcriptome wide analysis of gene expression and tail dynamics using end capture nanopore cDNA sequencing", "94bf5509-4622-4d5f-b7c5-6a14bdfac340", "Other", "RNA polyadenylation plays a central role in RNA maturation  fate  and stability. In response to developmental cues  polyA tail lengths can vary  affecting the translation efficiency and stability of mRNAs. Here  we develop Nanopore three prime end capture sequencing Nano3P seq  a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance  tail composition and tail length dynamics at per read resolution. By employing a template switching based sequencing protocol  Nano3P seq can sequence any given RNA molecule from its three prime end  regardless of its polyadenylation status  without xxx need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P seq captures a wide diversity of RNA biotypes  providing quantitative estimates of RNA abundance and tail lengths in mRNA  lncRNA  sn/snoRNA  scaRNA  and rRNA molecules. We find that  in addition to mRNA and lncRNA  polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover  we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform specific level  correlating with mRNA decay. Finally  we identify non A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis.  Overall  Nano3P seq is a simple and robust method for accurately estimating transcript levels  tail lengths  and tail composition heterogeneity in individual reads  with minimal library preparation biases  both in the coding and non coding transcriptome.", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", null, "Zebrafish Ribodepleted RNA 2hpf  4hpf  6hpf", "Zebrafish Ribodepleted Rep1", "SAMEA110100411", "CENTER FOR GENOMIC REGULATION (CRG)", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10|External Id:SAMEA110100411|INSDC center alias:CENTER FOR GENOMIC REGULATION CRG|INSDC center name:CENTER FOR GENOMIC REGULATION CRG|INSDC first public:2022 10 10T00:20:53Z|INSDC last update:2022 10 10T00:20:53Z|INSDC status:public|Submitter Id:Zebrafish Ribodepleted Rep1|common name:zebrafish|sample name:Zebrafish Ribodepleted Rep1", null, null, null, null, null, null, null, null, "MinION sequencing", "ena EXPERIMENT TAB 13 06 2022 16:07:52:807 813", "cDNA786327 ZFRDR1", "1", null, null, "RNA-Seq", "TRANSCRIPTOMIC", "other", "SINGLE", "OXFORD_NANOPORE", "MinION", null, "ERP138294", "MinION sequencing", "ENA FIRST PUBLIC:2022 10 10|ENA LAST UPDATE:2022 10 10", "cDNA786327_ZFRDR1.tar.gz", "nanopore", 1900613556.0, 1660167.0, "ena RUN TAB 13 06 2022 16:07:52:807 814", "0:1144.83", "A:473050820;C:438054520;G:425169743;T:564338473;N:0", 1144, null, null, null, 473050820, 438054520, 425169743, 564338473, 0, "ERX9385636", "ERS12199236", "ERA15547404", "CENTER FOR GENOMIC REGULATION (CRG)|European Nucleotide Archive", "CENTER FOR GENOMIC REGULATION (CRG)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, "ont", "ont", "3prime", "poly_a", "unknown", "bulk", "unknown", "unknown", null, "Spain", "2022-10-10", "Multi-stage", "Embryo", "Undetermined", "Embryo Imprecise"], [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"], [13299, "ERR984502", "ERX1065723", "ERS715232", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367580", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367580|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 24 sc 2286404|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCAGCTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 24 sc 2286404|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#24", "13741631", "Illumina sequencing of library 13741631  constructed from sample accession ERS715232 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCAGCTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#24.cram", "cram", 570705070.0, 4390039.0, "SC RUN 16164 8#24", "0:55 1:75", "A:167603013;C:88769000;G:138595187;T:175336986;N:400884", 55, 75, null, null, 167603013, 88769000, 138595187, 175336986, 400884, "ERX1065723", "ERS715232", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.18131, 0.44012, 0.12372, 0.1122, 0.96759, 0.93835, 0.63426, 0.57651, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13300, "ERR984501", "ERX1065722", "ERS715231", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367579", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367579|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 23 sc 2286403|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTAGTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 23 sc 2286403|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#23", "13741630", "Illumina sequencing of library 13741630  constructed from sample accession ERS715231 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACTAGTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#23.cram", "cram", 682831890.0, 5252553.0, "SC RUN 16164 8#23", "0:55 1:75", "A:201267036;C:106752351;G:152982998;T:221347859;N:481646", 55, 75, null, null, 201267036, 106752351, 152982998, 221347859, 481646, "ERX1065722", "ERS715231", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19992, 0.5315, 0.13001, 0.13817, 0.96025, 0.92261, 0.60527, 0.55787, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13301, "ERR984500", "ERX1065721", "ERS715230", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367578", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367578|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 22 sc 2286402|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGATTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 22 sc 2286402|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#22", "13741629", "Illumina sequencing of library 13741629  constructed from sample accession ERS715230 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCAGATTC.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#22.cram", "cram", 822730220.0, 6328694.0, "SC RUN 16164 8#22", "0:55 1:75", "A:241693794;C:130571512;G:177853465;T:272028770;N:582679", 55, 75, null, null, 241693794, 130571512, 177853465, 272028770, 582679, "ERX1065721", "ERS715230", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.2212, 0.54203, 0.15635, 0.13659, 0.96159, 0.9221, 0.59031, 0.55109, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13302, "ERR984499", "ERX1065720", "ERS715229", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367577", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367577|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 21 sc 2286401|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TATGCCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 21 sc 2286401|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#21", "13741628", "Illumina sequencing of library 13741628  constructed from sample accession ERS715229 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TATGCCAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#21.cram", "cram", 616663190.0, 4743563.0, "SC RUN 16164 8#21", "0:55 1:75", "A:176848806;C:99629161;G:137029831;T:202730142;N:425250", 55, 75, null, null, 176848806, 99629161, 137029831, 202730142, 425250, "ERX1065720", "ERS715229", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21864, 0.57174, 0.1516, 0.14103, 0.95905, 0.9192, 0.55513, 0.51633, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13303, "ERR984498", "ERX1065719", "ERS715228", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367576", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367576|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 20 sc 2286400|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGGCTCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 20 sc 2286400|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#20", "13741627", "Illumina sequencing of library 13741627  constructed from sample accession ERS715228 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGGCTCAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#20.cram", "cram", 708059950.0, 5446615.0, "SC RUN 16164 8#20", "0:55 1:75", "A:195264246;C:129715499;G:160155422;T:222438911;N:485872", 55, 75, null, null, 195264246, 129715499, 160155422, 222438911, 485872, "ERX1065719", "ERS715228", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19642, 0.48584, 0.11921, 0.11811, 0.95422, 0.91843, 0.62494, 0.56646, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13304, "ERR984497", "ERX1065718", "ERS715227", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367575", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367575|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 19 sc 2286399|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCATTGAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 19 sc 2286399|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#19", "13741626", "Illumina sequencing of library 13741626  constructed from sample accession ERS715227 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCATTGAG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#19.cram", "cram", 651943890.0, 5014953.0, "SC RUN 16164 8#19", "0:55 1:75", "A:191809575;C:111573276;G:144109125;T:204007845;N:444069", 55, 75, null, null, 191809575, 111573276, 144109125, 204007845, 444069, "ERX1065718", "ERS715227", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.17909, 0.44616, 0.12326, 0.12424, 0.96256, 0.93432, 0.56606, 0.52819, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13305, "ERR984496", "ERX1065717", "ERS715226", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367574", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367574|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 18 sc 2286398|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTATGCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 18 sc 2286398|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#18", "13741625", "Illumina sequencing of library 13741625  constructed from sample accession ERS715226 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGTATGCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#18.cram", "cram", 610476620.0, 4695974.0, "SC RUN 16164 8#18", "0:55 1:75", "A:181332724;C:99472616;G:132687929;T:196571397;N:411954", 55, 75, null, null, 181332724, 99472616, 132687929, 196571397, 411954, "ERX1065717", "ERS715226", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22636, 0.55559, 0.16123, 0.16819, 0.96025, 0.92967, 0.59576, 0.54685, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13306, "ERR984495", "ERX1065716", "ERS715225", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367573", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367573|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 17 sc 2286397|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCAGTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 17 sc 2286397|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#17", "13741624", "Illumina sequencing of library 13741624  constructed from sample accession ERS715225 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCCAGTCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#17.cram", "cram", 712320830.0, 5479391.0, "SC RUN 16164 8#17", "0:55 1:75", "A:204316194;C:115198795;G:158608369;T:233717227;N:480245", 55, 75, null, null, 204316194, 115198795, 158608369, 233717227, 480245, "ERX1065716", "ERS715225", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.2492, 0.59739, 0.17155, 0.14862, 0.95657, 0.92056, 0.58039, 0.53475, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13307, "ERR984494", "ERX1065715", "ERS715224", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367572", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367572|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 16 sc 2286396|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAAGTTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 16 sc 2286396|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#16", "13741623", "Illumina sequencing of library 13741623  constructed from sample accession ERS715224 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAAGTTCG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#16.cram", "cram", 793769340.0, 6105918.0, "SC RUN 16164 8#16", "0:55 1:75", "A:232264866;C:130235228;G:171709756;T:259013732;N:545758", 55, 75, null, null, 232264866, 130235228, 171709756, 259013732, 545758, "ERX1065715", "ERS715224", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22065, 0.5061, 0.15274, 0.13242, 0.95779, 0.92575, 0.57619, 0.53869, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13308, "ERR984493", "ERX1065714", "ERS715223", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367571", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367571|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 15 sc 2286395|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGGAGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 15 sc 2286395|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#15", "13741622", "Illumina sequencing of library 13741622  constructed from sample accession ERS715223 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCAGGAGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#15.cram", "cram", 785479760.0, 6042152.0, "SC RUN 16164 8#15", "0:55 1:75", "A:221631721;C:150326010;G:172964474;T:240036379;N:521176", 55, 75, null, null, 221631721, 150326010, 172964474, 240036379, 521176, "ERX1065714", "ERS715223", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.26761, 0.37751, 0.19858, 0.09301, 0.95162, 0.93129, 0.56282, 0.54289, 55, 75, "B", "B", "mate1-mate2 similar by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13309, "ERR984492", "ERX1065713", "ERS715222", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367570", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367570|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 14 sc 2286394|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCTCACGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 14 sc 2286394|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#14", "13741621", "Illumina sequencing of library 13741621  constructed from sample accession ERS715222 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCTCACGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#14.cram", "cram", 673293530.0, 5179181.0, "SC RUN 16164 8#14", "0:55 1:75", "A:188203068;C:115934699;G:155324541;T:213374769;N:456453", 55, 75, null, null, 188203068, 115934699, 155324541, 213374769, 456453, "ERX1065713", "ERS715222", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21855, 0.57586, 0.15197, 0.14501, 0.95946, 0.93077, 0.56453, 0.52351, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13310, "ERR984491", "ERX1065712", "ERS715221", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367569", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367569|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 13 sc 2286393|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTTCGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 13 sc 2286393|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#13", "13741620", "Illumina sequencing of library 13741620  constructed from sample accession ERS715221 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACTTCGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#13.cram", "cram", 783634020.0, 6027954.0, "SC RUN 16164 8#13", "0:55 1:75", "A:231435775;C:129417634;G:178939153;T:243302897;N:538561", 55, 75, null, null, 231435775, 129417634, 178939153, 243302897, 538561, "ERX1065712", "ERS715221", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20287, 0.52947, 0.14655, 0.13479, 0.96299, 0.93703, 0.56376, 0.52626, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13311, "ERR984490", "ERX1065711", "ERS715220", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367568", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367568|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 12 sc 2286392|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGAACTGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 12 sc 2286392|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#12", "13741619", "Illumina sequencing of library 13741619  constructed from sample accession ERS715220 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGAACTGG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#12.cram", "cram", 746397730.0, 5741521.0, "SC RUN 16164 8#12", "0:55 1:75", "A:217959769;C:123138989;G:158254272;T:246531930;N:512770", 55, 75, null, null, 217959769, 123138989, 158254272, 246531930, 512770, "ERX1065711", "ERS715220", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20695, 0.57674, 0.14123, 0.14978, 0.95899, 0.9138, 0.56705, 0.57522, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13312, "ERR984489", "ERX1065710", "ERS715219", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367567", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367567|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 11 sc 2286391|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTGGTATG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 11 sc 2286391|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#11", "13741618", "Illumina sequencing of library 13741618  constructed from sample accession ERS715219 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTGGTATG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#11.cram", "cram", 630077890.0, 4846753.0, "SC RUN 16164 8#11", "0:55 1:75", "A:181555459;C:107913967;G:131155798;T:209016157;N:436509", 55, 75, null, null, 181555459, 107913967, 131155798, 209016157, 436509, "ERX1065710", "ERS715219", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.23034, 0.58664, 0.14994, 0.16031, 0.9517, 0.9134, 0.43491, 0.55537, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13313, "ERR984488", "ERX1065709", "ERS715218", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367566", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:57Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367566|INSDC center name:SC|INSDC first public:2015 08 18T09:04:57Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 10 sc 2286390|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAACGCTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 10 sc 2286390|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#10", "13741617", "Illumina sequencing of library 13741617  constructed from sample accession ERS715218 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAACGCTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#10.cram", "cram", 522425410.0, 4018657.0, "SC RUN 16164 8#10", "0:55 1:75", "A:136789388;C:88820863;G:131305976;T:165156817;N:352366", 55, 75, null, null, 136789388, 88820863, 131305976, 165156817, 352366, "ERX1065709", "ERS715218", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.16976, 0.61187, 0.10677, 0.16777, 0.96096, 0.93093, 0.59603, 0.58893, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13314, "ERR984487", "ERX1065708", "ERS715217", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367565", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367565|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 9 sc 2286389|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCGAAGTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 9 sc 2286389|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#9", "13741616", "Illumina sequencing of library 13741616  constructed from sample accession ERS715217 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCGAAGTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#9.cram", "cram", 662372100.0, 5095170.0, "SC RUN 16164 8#9", "0:55 1:75", "A:186346528;C:114321445;G:137139385;T:224106023;N:458719", 55, 75, null, null, 186346528, 114321445, 137139385, 224106023, 458719, "ERX1065708", "ERS715217", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22854, 0.63729, 0.14734, 0.16003, 0.95089, 0.90751, 0.51773, 0.50977, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13315, "ERR984486", "ERX1065707", "ERS715216", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367564", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367564|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 8 sc 2286388|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCATTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 8 sc 2286388|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#8", "13741615", "Illumina sequencing of library 13741615  constructed from sample accession ERS715216 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCCATTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#8.cram", "cram", 857007060.0, 6592362.0, "SC RUN 16164 8#8", "0:55 1:75", "A:257958701;C:131661477;G:190276240;T:276500331;N:610311", 55, 75, null, null, 257958701, 131661477, 190276240, 276500331, 610311, "ERX1065707", "ERS715216", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21266, 0.595, 0.14225, 0.14037, 0.95964, 0.92232, 0.54595, 0.53111, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13316, "ERR984485", "ERX1065706", "ERS715215", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367563", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367563|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 7 sc 2286387|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTCTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 7 sc 2286387|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#7", "13741614", "Illumina sequencing of library 13741614  constructed from sample accession ERS715215 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAGTCTTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#7.cram", "cram", 742942590.0, 5714943.0, "SC RUN 16164 8#7", "0:55 1:75", "A:228799118;C:117009382;G:166056205;T:230565934;N:511951", 55, 75, null, null, 228799118, 117009382, 166056205, 230565934, 511951, "ERX1065706", "ERS715215", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.19264, 0.50767, 0.1428, 0.15173, 0.9666, 0.94606, 0.57413, 0.53902, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13317, "ERR984484", "ERX1065705", "ERS715214", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367562", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367562|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 6 sc 2286386|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTGGTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 6 sc 2286386|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#6", "13741613", "Illumina sequencing of library 13741613  constructed from sample accession ERS715214 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGTGGTTG.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#6.cram", "cram", 908951420.0, 6991934.0, "SC RUN 16164 8#6", "0:55 1:75", "A:266807632;C:158160652;G:194742499;T:288619707;N:620930", 55, 75, null, null, 266807632, 158160652, 194742499, 288619707, 620930, "ERX1065705", "ERS715214", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20021, 0.52069, 0.13208, 0.1197, 0.95775, 0.93324, 0.52946, 0.52456, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13318, "ERR984483", "ERX1065704", "ERS715213", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367561", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367561|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 5 sc 2286385|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCTCAAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 5 sc 2286385|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#5", "13741612", "Illumina sequencing of library 13741612  constructed from sample accession ERS715213 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TCCTCAAT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#5.cram", "cram", 771659330.0, 5935841.0, "SC RUN 16164 8#5", "0:55 1:75", "A:225161011;C:121434126;G:174639826;T:249876341;N:548026", 55, 75, null, null, 225161011, 121434126, 174639826, 249876341, 548026, "ERX1065704", "ERS715213", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21069, 0.57513, 0.14228, 0.12501, 0.95672, 0.92488, 0.54397, 0.52111, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13319, "ERR984482", "ERX1065703", "ERS715212", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367560", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367560|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 4 sc 2286384|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACAGGAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 4 sc 2286384|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#4", "13741611", "Illumina sequencing of library 13741611  constructed from sample accession ERS715212 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TACAGGAT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#4.cram", "cram", 784183790.0, 6032183.0, "SC RUN 16164 8#4", "0:55 1:75", "A:219327080;C:128313123;G:169475262;T:266538585;N:529740", 55, 75, null, null, 219327080, 128313123, 169475262, 266538585, 529740, "ERX1065703", "ERS715212", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.20392, 0.58702, 0.12804, 0.12156, 0.96047, 0.9133, 0.61438, 0.53735, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13320, "ERR984481", "ERX1065702", "ERS715211", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367559", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367559|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 3 sc 2286383|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTGACT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 3 sc 2286383|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#3", "13741610", "Illumina sequencing of library 13741610  constructed from sample accession ERS715211 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TAGTGACT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#3.cram", "cram", 810265170.0, 6232809.0, "SC RUN 16164 8#3", "0:55 1:75", "A:237117705;C:130774361;G:170817877;T:270997878;N:557349", 55, 75, null, null, 237117705, 130774361, 170817877, 270997878, 557349, "ERX1065702", "ERS715211", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.22414, 0.5931, 0.14561, 0.13304, 0.95651, 0.91423, 0.59465, 0.54152, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13321, "ERR984480", "ERX1065701", "ERS715210", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367558", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367558|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 2 sc 2286382|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCTGCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 2 sc 2286382|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#2", "13741609", "Illumina sequencing of library 13741609  constructed from sample accession ERS715210 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TTCCTGCT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#2.cram", "cram", 698194770.0, 5370729.0, "SC RUN 16164 8#2", "0:55 1:75", "A:190784194;C:110138446;G:188418516;T:208362798;N:490816", 55, 75, null, null, 190784194, 110138446, 188418516, 208362798, 490816, "ERX1065701", "ERS715210", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.17398, 0.58306, 0.10399, 0.12476, 0.96372, 0.94468, 0.64309, 0.54721, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [13322, "ERR984479", "ERX1065700", "ERS715209", "ERP006132", "PRJEB6584", "Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project", "Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191", "Transcriptome Analysis", "Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.", null, null, null, null, "SAMEA3367557", "SC", "ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028   Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367557|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 1 sc 2286381|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna  allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGCGATCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 1 sc 2286381|scientific name:Danio rerio", null, null, null, null, null, null, null, null, "Illumina HiSeq 2000 paired end sequencing", "SC EXP 16164 8#1", "13741608", "Illumina sequencing of library 13741608  constructed from sample accession ERS715209 for study accession ERP006132.  This is part of an Illumina multiplexed sequencing run 16164 8.  This submission includes reads tagged with the sequence TGCGATCT.", "Transcriptome counting qPCR only", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2000", null, "ERP006132", "Illumina HiSeq 2000 paired end sequencing", "ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16", "16164_8#1.cram", "cram", 882032060.0, 6784862.0, "SC RUN 16164 8#1", "0:55 1:75", "A:258098215;C:144707482;G:194402683;T:284218124;N:605556", 55, 75, null, null, 258098215, 144707482, 194402683, 284218124, 605556, "ERX1065700", "ERS715209", "ERA466416", "The Wellcome Trust Sanger Institute|European Nucleotide Archive", "Wellcome Sanger Institute", 2, 0.21585, 0.55375, 0.14409, 0.13461, 0.96015, 0.9246, 0.61791, 0.5495, 55, 75, "T", "B", "mate1 technical by mapping diff", "illumina", "hiseq_era", "3prime", "cdna_unspecified", "unknown", "bulk", "unknown", "unknown", null, "United Kingdom", "2015-08-18", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [24927, "SRR25557778", "SRX21286665", "SRS18536778", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant XI", "GSM7688794", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant XI", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688794", "GSM7688794: Morphant XI; Danio rerio; RNA Seq", "GSM7688794 r1", "GSM7688794", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-XI_S7_L001_R1_001.fastq.gz", "fastq", 420092501.0, 5668059.0, "GSM7688794 r1", "0:74.12", "A:113006440;C:96725832;G:99320773;T:110915967;N:123489", 74, null, null, null, 113006440, 96725832, 99320773, 110915967, 123489, "SRX21286665", "SRS18536778", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94757, null, 0.06229, null, 0.72364, null, 0.46676, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24928, "SRR25557779", "SRX21286665", "SRS18536778", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant XI", "GSM7688794", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant XI", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688794", "GSM7688794: Morphant XI; Danio rerio; RNA Seq", "GSM7688794 r1", "GSM7688794", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-XI_S7_L002_R1_001.fastq.gz", "fastq", 421869780.0, 5690053.0, "GSM7688794 r2", "0:74.14", "A:113530489;C:97144227;G:99697959;T:111388539;N:108566", 74, null, null, null, 113530489, 97144227, 99697959, 111388539, 108566, "SRX21286665", "SRS18536778", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94921, null, 0.06226, null, 0.72462, null, 0.4738, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24929, "SRR25557780", "SRX21286665", "SRS18536778", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant XI", "GSM7688794", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant XI", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688794", "GSM7688794: Morphant XI; Danio rerio; RNA Seq", "GSM7688794 r1", "GSM7688794", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-XI_S7_L003_R1_001.fastq.gz", "fastq", 425064659.0, 5734041.0, "GSM7688794 r3", "0:74.13", "A:114342253;C:97873100;G:100532599;T:112196433;N:120274", 74, null, null, null, 114342253, 97873100, 100532599, 112196433, 120274, "SRX21286665", "SRS18536778", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94886, null, 0.06112, null, 0.72425, null, 0.47055, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24930, "SRR25557781", "SRX21286665", "SRS18536778", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant XI", "GSM7688794", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant XI", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688794", "GSM7688794: Morphant XI; Danio rerio; RNA Seq", "GSM7688794 r1", "GSM7688794", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-XI_S7_L004_R1_001.fastq.gz", "fastq", 416990548.0, 5624902.0, "GSM7688794 r4", "0:74.13", "A:112153356;C:96009158;G:98613145;T:110097476;N:117413", 74, null, null, null, 112153356, 96009158, 98613145, 110097476, 117413, "SRX21286665", "SRS18536778", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94869, null, 0.06229, null, 0.72506, null, 0.47222, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24931, "SRR25557782", "SRX21286664", "SRS18536777", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant X", "GSM7688793", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant X", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688793", "GSM7688793: Morphant X; Danio rerio; RNA Seq", "GSM7688793 r1", "GSM7688793", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-X_S6_L001_R1_001.fastq.gz", "fastq", 434485284.0, 5881416.0, "GSM7688793 r1", "0:73.87", "A:116640351;C:100176557;G:102659919;T:114799536;N:208921", 73, null, null, null, 116640351, 100176557, 102659919, 114799536, 208921, "SRX21286664", "SRS18536777", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94533, null, 0.07176, null, 0.72464, null, 0.47632, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24932, "SRR25557783", "SRX21286664", "SRS18536777", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant X", "GSM7688793", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant X", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688793", "GSM7688793: Morphant X; Danio rerio; RNA Seq", "GSM7688793 r1", "GSM7688793", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-X_S6_L002_R1_001.fastq.gz", "fastq", 435203869.0, 5886556.0, "GSM7688793 r2", "0:73.93", "A:116869356;C:100363580;G:102830644;T:114973504;N:166785", 73, null, null, null, 116869356, 100363580, 102830644, 114973504, 166785, "SRX21286664", "SRS18536777", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94542, null, 0.07203, null, 0.72421, null, 0.47733, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24933, "SRR25557784", "SRX21286664", "SRS18536777", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant X", "GSM7688793", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant X", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688793", "GSM7688793: Morphant X; Danio rerio; RNA Seq", "GSM7688793 r1", "GSM7688793", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-X_S6_L003_R1_001.fastq.gz", "fastq", 439897269.0, 5951878.0, "GSM7688793 r3", "0:73.91", "A:118101648;C:101426657;G:103990006;T:116184480;N:194478", 73, null, null, null, 118101648, 101426657, 103990006, 116184480, 194478, "SRX21286664", "SRS18536777", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94486, null, 0.07224, null, 0.72448, null, 0.47915, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24934, "SRR25557785", "SRX21286664", "SRS18536777", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant X", "GSM7688793", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant X", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688793", "GSM7688793: Morphant X; Danio rerio; RNA Seq", "GSM7688793 r1", "GSM7688793", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-X_S6_L004_R1_001.fastq.gz", "fastq", 431385256.0, 5836029.0, "GSM7688793 r4", "0:73.92", "A:115804970;C:99459059;G:101962932;T:113975935;N:182360", 73, null, null, null, 115804970, 99459059, 101962932, 113975935, 182360, "SRX21286664", "SRS18536777", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94432, null, 0.07229, null, 0.7261, null, 0.47463, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24935, "SRR25557786", "SRX21286663", "SRS18536776", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant IX", "GSM7688792", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant IX", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688792", "GSM7688792: Morphant IX; Danio rerio; RNA Seq", "GSM7688792 r1", "GSM7688792", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-IX_S16_L001_R1_001.fastq.gz", "fastq", 513309395.0, 6929648.0, "GSM7688792 r1", "0:74.07", "A:137428462;C:118688804;G:122019962;T:134991729;N:180438", 74, null, null, null, 137428462, 118688804, 122019962, 134991729, 180438, "SRX21286663", "SRS18536776", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94188, null, 0.07029, null, 0.73772, null, 0.47692, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24936, "SRR25557787", "SRX21286663", "SRS18536776", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant IX", "GSM7688792", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant IX", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688792", "GSM7688792: Morphant IX; Danio rerio; RNA Seq", "GSM7688792 r1", "GSM7688792", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-IX_S16_L002_R1_001.fastq.gz", "fastq", 517356735.0, 6982196.0, "GSM7688792 r2", "0:74.10", "A:138524037;C:119650852;G:122965491;T:136056171;N:160184", 74, null, null, null, 138524037, 119650852, 122965491, 136056171, 160184, "SRX21286663", "SRS18536776", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94281, null, 0.06967, null, 0.73963, null, 0.48142, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24937, "SRR25557788", "SRX21286663", "SRS18536776", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant IX", "GSM7688792", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant IX", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688792", "GSM7688792: Morphant IX; Danio rerio; RNA Seq", "GSM7688792 r1", "GSM7688792", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-IX_S16_L003_R1_001.fastq.gz", "fastq", 519422328.0, 7010631.0, "GSM7688792 r3", "0:74.09", "A:139040684;C:120128748;G:123529198;T:136551898;N:171800", 74, null, null, null, 139040684, 120128748, 123529198, 136551898, 171800, "SRX21286663", "SRS18536776", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94297, null, 0.06942, null, 0.73726, null, 0.47499, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24938, "SRR25557789", "SRX21286663", "SRS18536776", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant IX", "GSM7688792", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant IX", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688792", "GSM7688792: Morphant IX; Danio rerio; RNA Seq", "GSM7688792 r1", "GSM7688792", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-IX_S16_L004_R1_001.fastq.gz", "fastq", 511640294.0, 6905748.0, "GSM7688792 r4", "0:74.09", "A:136914638;C:118302866;G:121704665;T:134543505;N:174620", 74, null, null, null, 136914638, 118302866, 121704665, 134543505, 174620, "SRX21286663", "SRS18536776", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94176, null, 0.07029, null, 0.73868, null, 0.47987, null, 74, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24939, "SRR25557790", "SRX21286662", "SRS18536775", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant VIII", "GSM7688791", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant VIII", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688791", "GSM7688791: Morphant VIII; Danio rerio; RNA Seq", "GSM7688791 r1", "GSM7688791", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-VIII_S14_L001_R1_001.fastq.gz", "fastq", 429446821.0, 5803178.0, "GSM7688791 r1", "0:74.00", "A:114793535;C:99506379;G:102226914;T:112748761;N:171232", 74, null, null, null, 114793535, 99506379, 102226914, 112748761, 171232, "SRX21286662", "SRS18536775", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94175, null, 0.06669, null, 0.73673, null, 0.48179, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24940, "SRR25557791", "SRX21286662", "SRS18536775", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant VIII", "GSM7688791", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant VIII", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688791", "GSM7688791: Morphant VIII; Danio rerio; RNA Seq", "GSM7688791 r1", "GSM7688791", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-VIII_S14_L002_R1_001.fastq.gz", "fastq", 434629893.0, 5870828.0, "GSM7688791 r2", "0:74.03", "A:116216940;C:100703527;G:103463365;T:114092681;N:153380", 74, null, null, null, 116216940, 100703527, 103463365, 114092681, 153380, "SRX21286662", "SRS18536775", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94143, null, 0.0676, null, 0.73791, null, 0.48091, null, 75, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"], [24941, "SRR25557792", "SRX21286662", "SRS18536775", "SRP453884", "PRJNA1003026", "Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [bulk RNA Seq]", "GSE240238", "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: Ten samples were analysed in total  all at 27 hpf. Five biological repicates were performed for V1 and dI2 spinal interneurons from uninjected wild type control embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background. Five biological replicates were performed for V1 and dI2 spinal interneurons from hmx2;hmx3a double knock down DKD morphant embryos in the Tghmx CNEIII:cfos:Gal4 VP16 UAS:EGFPSU41 background.", "parent bioproject:PRJNA1003022", "pubmed:38017520", null, "Morphant VIII", "GSM7688791", null, "source name:Spinal Cord|tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant|geo loc name:missing|collection date:missing", "Morphant VIII", "We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. We trimmed the adapter sequence \u201cCTGTCTCTTATACACATCT\u201d from the 3\u2019 end using default parameters  before trimming bases from the 5\u2019 end  selecting an end minimum quality value Phred score of 32  and a minimum read length of 65 bases. We aligned reads using default parameters and the STAR 2.6.1d algorithm. We normalized the log expression ratios using a Trimmed Means of M values TMM weighted algorithm. We performed differential expression analysis using the Gene Specific Analysis GSA algorithm in Partek Flow. The outcome of GSA was assessed by hierarchical clustering heatmap plotting  clustering by features  using average linkage and Euclidean cluster distance and point distance metrics respectively. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated differential expression analysis file comparing all uninjected control samples versus all hmx2;hmx3a DKD morphant embryos.", "Spinal Cord", "The hmx2;hmx3a DKD morphant embryos used in this study were obtained by injecting 3.5 nl of a mixture containing 2 ng/nl each of a translation blocking hmx2 morpholino 5\u2019 TTCCGCTGTCCTCCGAATTATTCAT and a translation blocking hmx3a morpholino 5\u2019 ACGTATCCTGTGTTGTTTCGGGCAT  plus 5 ng/nl of a control zebrafish p53 morpholino 5\u2019 GCGCCATTGCTTTGCAAGAATTG  into the single cell of a one cell stage Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 embryo all morpholinos obtained from Gene Tools.   Morpholino injections always produce a spectrum of phenotypes  since it is hard to ensure that every cell receives the same dose. Therefore  prior to processing for FACS at 27 hpf  we removed any embryos with severely abnormal morphology stunted length and/or severely developmentally delayed  likely caused by receiving too much morpholino. DKD morphant embryos display a slight curled tail down morphology. Embryos that lacked this morphology and may therefore not have received any or sufficient morpholino were also removed before processing for FACS.", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer\u2019s instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", "The hmx2;hmx3a double knockdown DKD morphant embryos used in this study exhibit delayed development from somitogenesis stages onwards when compared to uninjected controls. To circumvent this  they were incubated at 32oC from 9 hpf onwards. This ensured that control and injected embryos reached the desired developmental stage of 27 hpf at approximately the same time. The lateral line primordium does not migrate in DKD animals  so this could not be used to stage injected embryos. Instead  these embryos were visually inspected and processed for fluorescence activated cell sorting FACS when they displayed the same head trunk angle  head size and eye size as prim staged uninjected control embryos.", "tissue:Spinal Cord|cell line:Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41|cell type:V1 and dI2 spinal interneurons|genotype:hmx2;hmx3a double knockdowm morphant|treatment:hmx2;hmx3a double knockdowm morphant", "GSM7688791", "GSM7688791: Morphant VIII; Danio rerio; RNA Seq", "GSM7688791 r1", "GSM7688791", "1", "Uninjected control embryos and hmx2;hmx3a DKD morphant embryos in the Tghmx CNEIII:cfos:GAL4 VP16 UAS:EGFPSU41 background generated as described above were screened for fluorescence from 24 hpf onwards. Only EGFP positive control and hmx2;hmx3a DKD morphant animals were used for dissociation and fluorescent activated cell sorting FACS at 27 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 30 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. Cells were sorted directly in to sterile 1.5 ml microcentrifuge tubes containing 100 \u00b5l of Buffer RLT Qiagen RNeasy Micro Kit  74004 plus 143 mM\uf020\u03b2 mercaptoethanol. Sorted cells were stored at  80oC prior to RNA extraction.  Frozen FAC sorted cell lysates were removed from storage at  80oC and thawed in a 37oC waterbath  before transferring to sterile microcentrifuge tubes. If necessary  sample volumes were completed to 250 \u00b5l with UltraPure DNase/RNase Free distilled water ThermoFisher Scientific  10977035. 750 \u00b5l TRIzol LS Reagent ThermoFisher Scientific  10296028 was added to each 250 \u00b5l sample  before homogenising by gently pipetting up and down ten times with a sterile p1000 pipette tip. Samples were immediately transferred to Phasemaker tubes which had been pre centrifuged as per the manufacturer's instructions ThermoFisher Scientific  A33248  before incubating for 5 minutes at room temperature. 200 \u00b5l chloroform was added to each sample. The tubes were then shaken vigorously for 15 seconds and incubated for a further 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 16 000 x g at 4oC  before transferring the RNA containing upper aqueous phase to a sterile centrifuge tube and adding one volume of 70% RNase free ethanol. Samples were inverted to mix thoroughly  and the supernatant immediately loaded to an RNEasy MinElute column from the RNeasy Micro Kit  Qiagen  74004  before centrifuging for 15 seconds at 10 000 rpm. Wash steps with RW1 buffer  RPE buffer and 80% RNase free ethanol was performed as per the RNeasy Micro Kit instructions. Samples were eluted in 14 \u00b5l RNase free water. RNA integrity was assessed with the Agilent RNA 6000 Pico chip Agilent  5067 1513 on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity RIN values >9 were used for library preparation. RNA concentrations were measured with the Qubit RNA High Sensitivity Assay Kit ThermoFisher Scientific  Q32852 and a Qubit 3.0 fluorometer ThermoFisher Scientific  Q33216. cDNA was synthesised using the SMART Seq v4 Ultra Low Input RNA Kit for Sequencing Takara  634888  and used to make sequencing libraries with the Nextera XT DNA Library Preparation Kit Illumina  FC 131 1024. cDNA and library quality were measured with the Agilent High Sensitivity DNA Kit Agilent  5067 4626 on an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina NextSeq500 to a depth of 20 million reads per sample Illumina NextSeq 500/500 High Output Kit  v2.5  75 cycles  20024906.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "SINGLE", "ILLUMINA", "NextSeq 500", null, "SRP453884", null, "loader:fastq load.py", "Morphant-VIII_S14_L003_R1_001.fastq.gz", "fastq", 435879881.0, 5888685.0, "GSM7688791 r3", "0:74.02", "A:116548094;C:100971153;G:103794902;T:114400770;N:164962", 74, null, null, null, 116548094, 100971153, 103794902, 114400770, 164962, "SRX21286662", "SRS18536775", "SRA1688461", "Lewis Lab, Biology, Syracuse University", "Lewis Lab, Biology, Syracuse University", 1, 0.94195, null, 0.06686, null, 0.73785, null, 0.48044, null, 73, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2023-08-07", "Multi-stage", "Embryo", "Spinal Cord", "Nervous System"]], "truncated": false, "filtered_table_rows_count": 2090, "expanded_columns": [], "expandable_columns": [], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": [], "units": {}, "query": {"sql": "select rowid, [run.accession], [experiment.accession], [sample.accession], [study.accession], bioproject, [study.title], [study.alias], [study.type], [study.abstract], [study.attributes], [study.PMIDs], [sample.description], [sample.title], [sample.alias], [sample.centername], [sample.attributes], [GEOsample.title], [GEOsample.dataprocessing], [GEOsample.source], [GEOsample.treatmentprotocol], [GEOsample.extractprotocol], [GEOsample.growthprotocol], [GEOsample.characteristics], [GEOsample.accession], [experiment.title], [experiment.alias], [experiment.library_name], [experiment.design_description], [experiment.library_construction_protocol], [experiment.attributes], [experiment.library_strategy], [experiment.library_source], [experiment.library_selection], [experiment.library_layout], [experiment.platform], [experiment.instrument_model], [experiment.spot_descriptor], [experiment.study_ref], [run.title], [run.attributes], [run.filename], [run.semantic_name], [run.total_bases], [run.total_spots], [run.alias], [run.read_lengths], [run.base_counts], [run.r1_length], [run.r2_length], [run.r3_length], [run.r4_length], [run.Acount], [run.Ccount], [run.Gcount], [run.Tcount], [run.Ncount], [run.experiment], [run.pool_member], [submission.accession], [submission.srasource], [submission.bioprojectsource], [seqdetective.n_mates], [seqdetective.mapping_rate.mate1], [seqdetective.mapping_rate.mate2], [seqdetective.nofeature_rate.mate1], [seqdetective.nofeature_rate.mate2], [seqdetective.sparsity.mate1], [seqdetective.sparsity.mate2], [seqdetective.pos_strand_rate.mate1], [seqdetective.pos_strand_rate.mate2], [seqdetective.readlen.mate1], [seqdetective.readlen.mate2], [seqdetective.judgement.mate1], [seqdetective.judgement.mate2], 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