{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.platform = \"ILLUMINA\", technology = \"10x\" and tissue_curation = \"Thymus\"", "rows": [[67584, "SRR17218121", "SRX13397710", "SRS11300870", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 4", "GSM5732082", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 4", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732082", "GSM5732082: Juvenile Thymus 4; Danio rerio; RNA Seq", "GSM5732082", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. 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Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 4", "GSM5732082", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 4", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732082", "GSM5732082: Juvenile Thymus 4; Danio rerio; RNA Seq", "GSM5732082", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732082", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_6_S4_L002_I1_001.fastq.gz 4wpfThymus_6_S4_L002_R1_001.fastq.gz 4wpfThymus_6_S4_L002_R2_001.fastq.gz", "fastq fastq fastq", 14660763541.0, 115439083.0, "GSM5732082 r2", "0:8 1:28 2:91", "A:3154820274;C:2128697452;G:2532561232;T:2688755742;N:121853", 8, 28, 91, null, 3154820274, 2128697452, 2532561232, 2688755742, 121853, "SRX13397710", "SRS11300870", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.87166, null, 0.22046, null, 0.8407, null, 0.5334, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67586, "SRR17218119", "SRX13397709", "SRS11300871", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 3", "GSM5732081", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 3", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732081", "GSM5732081: Juvenile Thymus 3; Danio rerio; RNA Seq", "GSM5732081", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732081", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_5_S3_L001_I1_001.fastq.gz 4wpfThymus_5_S3_L001_R1_001.fastq.gz 4wpfThymus_5_S3_L001_R2_001.fastq.gz", "fastq fastq fastq", 14488701909.0, 114084267.0, "GSM5732081 r1", "0:8 1:28 2:91", "A:3107408243;C:2119850736;G:2503503910;T:2650750983;N:154425", 8, 28, 91, null, 3107408243, 2119850736, 2503503910, 2650750983, 154425, "SRX13397709", "SRS11300871", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.85944, null, 0.20997, null, 0.84064, null, 0.53634, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67587, "SRR17218120", "SRX13397709", "SRS11300871", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 3", "GSM5732081", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 3", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732081", "GSM5732081: Juvenile Thymus 3; Danio rerio; RNA Seq", "GSM5732081", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. 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The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732081", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_5_S3_L002_I1_001.fastq.gz 4wpfThymus_5_S3_L002_R1_001.fastq.gz 4wpfThymus_5_S3_L002_R2_001.fastq.gz", "fastq fastq fastq", 14352524508.0, 113012004.0, "GSM5732081 r2", "0:8 1:28 2:91", "A:3079316058;C:2096769810;G:2479668921;T:2628218918;N:118657", 8, 28, 91, null, 3079316058, 2096769810, 2479668921, 2628218918, 118657, "SRX13397709", "SRS11300871", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.85981, null, 0.21085, null, 0.8393, null, 0.52667, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67588, "SRR17218117", "SRX13397708", "SRS11300869", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 2", "GSM5732080", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 2", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732080", "GSM5732080: Juvenile Thymus 2; Danio rerio; RNA Seq", "GSM5732080", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732080", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_3_S2_L001_I1_001.fastq.gz 4wpfThymus_3_S2_L001_R1_001.fastq.gz 4wpfThymus_3_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 17459416821.0, 137475723.0, "GSM5732080 r1", "0:8 1:28 2:91", "A:3740652508;C:2562291967;G:3023715848;T:3183446304;N:184166", 8, 28, 91, null, 3740652508, 2562291967, 3023715848, 3183446304, 184166, "SRX13397708", "SRS11300869", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.84416, null, 0.21191, null, 0.83897, null, 0.53108, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67589, "SRR17218118", "SRX13397708", "SRS11300869", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 2", "GSM5732080", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 2", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732080", "GSM5732080: Juvenile Thymus 2; Danio rerio; RNA Seq", "GSM5732080", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732080", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_3_S2_L002_I1_001.fastq.gz 4wpfThymus_3_S2_L002_R1_001.fastq.gz 4wpfThymus_3_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 17241251014.0, 135757882.0, "GSM5732080 r2", "0:8 1:28 2:91", "A:3695117374;C:2526230853;G:2985988000;T:3146489343;N:141692", 8, 28, 91, null, 3695117374, 2526230853, 2985988000, 3146489343, 141692, "SRX13397708", "SRS11300869", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.84569, null, 0.21301, null, 0.83899, null, 0.53404, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67590, "SRR17218115", "SRX13397707", "SRS11300868", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 1", "GSM5732079", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 1", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732079", "GSM5732079: Juvenile Thymus 1; Danio rerio; RNA Seq", "GSM5732079", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732079", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_1_S1_L001_I1_001.fastq.gz 4wpfThymus_1_S1_L001_R1_001.fastq.gz 4wpfThymus_1_S1_L001_R2_001.fastq.gz", "fastq fastq fastq", 16221392627.0, 127727501.0, "GSM5732079 r1", "0:8 1:28 2:91", "A:3503038095;C:2359078159;G:2792714907;T:2968200161;N:171269", 8, 28, 91, null, 3503038095, 2359078159, 2792714907, 2968200161, 171269, "SRX13397707", "SRS11300868", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.84501, null, 0.21705, null, 0.8379, null, 0.53566, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67591, "SRR17218116", "SRX13397707", "SRS11300868", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Juvenile Thymus 1", "GSM5732079", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Juvenile Thymus 1", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Juvenile|age:4 wpf bleed:no|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732079", "GSM5732079: Juvenile Thymus 1; Danio rerio; RNA Seq", "GSM5732079", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732079", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "4wpfThymus_1_S1_L002_I1_001.fastq.gz 4wpfThymus_1_S1_L002_R1_001.fastq.gz 4wpfThymus_1_S1_L002_R2_001.fastq.gz", "fastq fastq fastq", 16074886951.0, 126573913.0, "GSM5732079 r2", "0:8 1:28 2:91", "A:3472769520;C:2334135014;G:2767323491;T:2943866297;N:131761", 8, 28, 91, null, 3472769520, 2334135014, 2767323491, 2943866297, 131761, "SRX13397707", "SRS11300868", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.84624, null, 0.21812, null, 0.83918, null, 0.52823, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Multi-stage", "Multi-stage", "Thymus", "Hematopoietic System"], [67592, "SRR17218113", "SRX13397706", "SRS11300867", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 2", "GSM5732078", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 2", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732078", "GSM5732078: Adult Thymus 2; Danio rerio; RNA Seq", "GSM5732078", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732078", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "lck-eGFP_Thymus_2__S4_L001_I1_001.fastq.gz lck-eGFP_Thymus_2__S4_L001_R1_001.fastq.gz lck-eGFP_Thymus_2__S4_L001_R2_001.fastq.gz", "fastq fastq fastq", 13928486651.0, 110400658.0, "GSM5732078 r1", "0:8 1:27.57 2:90.59", "A:2982389810;C:2043569636;G:2322019641;T:2652821180;N:252711", 8, 27, 90, null, 2982389810, 2043569636, 2322019641, 2652821180, 252711, "SRX13397706", "SRS11300867", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.9102, null, 0.22477, null, 0.80227, null, 0.51027, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67593, "SRR17218114", "SRX13397706", "SRS11300867", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 2", "GSM5732078", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 2", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732078", "GSM5732078: Adult Thymus 2; Danio rerio; RNA Seq", "GSM5732078", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732078", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "lck-eGFP_Thymus_2__S4_L002_I1_001.fastq.gz lck-eGFP_Thymus_2__S4_L002_R1_001.fastq.gz lck-eGFP_Thymus_2__S4_L002_R2_001.fastq.gz", "fastq fastq fastq", 13882495233.0, 110035762.0, "GSM5732078 r2", "0:8 1:27.57 2:90.59", "A:2973325031;C:2035977607;G:2312724405;T:2645711786;N:286217", 8, 27, 90, null, 2973325031, 2035977607, 2312724405, 2645711786, 286217, "SRX13397706", "SRS11300867", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.9095, null, 0.22389, null, 0.80135, null, 0.52051, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67594, "SRR17218111", "SRX13397705", "SRS11300865", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 1", "GSM5732077", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 1", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732077", "GSM5732077: Adult Thymus 1; Danio rerio; RNA Seq", "GSM5732077", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732077", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "lck-eGFP_Thymus_1__S4_L001_I1_001.fastq.gz lck-eGFP_Thymus_1__S4_L001_R1_001.fastq.gz lck-eGFP_Thymus_1__S4_L001_R2_001.fastq.gz", "fastq fastq fastq", 13683556018.0, 108451992.0, "GSM5732077 r1", "0:8 1:27.57 2:90.60", "A:2970924288;C:1965313899;G:2264518373;T:2624238930;N:636831", 8, 27, 90, null, 2970924288, 1965313899, 2264518373, 2624238930, 636831, "SRX13397705", "SRS11300865", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.91685, null, 0.27522, null, 0.81105, null, 0.52662, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67595, "SRR17218112", "SRX13397705", "SRS11300865", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 1", "GSM5732077", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 1", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:Tglck:eGFP|developmental stage:Adult|age:3 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732077", "GSM5732077: Adult Thymus 1; Danio rerio; RNA Seq", "GSM5732077", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732077", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "lck-eGFP_Thymus_1__S4_L002_I1_001.fastq.gz lck-eGFP_Thymus_1__S4_L002_R1_001.fastq.gz lck-eGFP_Thymus_1__S4_L002_R2_001.fastq.gz", "fastq fastq fastq", 13739084909.0, 108892246.0, "GSM5732077 r2", "0:8 1:27.57 2:90.60", "A:2986926683;C:1971756062;G:2268315286;T:2638089379;N:417199", 8, 27, 90, null, 2986926683, 1971756062, 2268315286, 2638089379, 417199, "SRX13397705", "SRS11300865", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.91881, null, 0.27676, null, 0.81217, null, 0.5208, null, 91, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67608, "SRR17218097", "SRX13397698", "SRS11300859", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 4", "GSM5732070", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 4", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732070", "GSM5732070: Adult Thymus 4; Danio rerio; RNA Seq", "GSM5732070", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732070", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "Fish_24_Thymus__S3_L001_I1_001.fastq.gz Fish_24_Thymus__S3_L001_R1_001.fastq.gz Fish_24_Thymus__S3_L001_R2_001.fastq.gz", "fastq fastq fastq", 18278645709.0, 144870332.0, "GSM5732070 r1", "0:8 1:27.57 2:90.60", "A:3985081516;C:2643040166;G:3063178524;T:3433439412;N:330251", 8, 27, 90, null, 3985081516, 2643040166, 3063178524, 3433439412, 330251, "SRX13397698", "SRS11300859", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.92193, null, 0.26629, null, 0.81191, null, 0.5476, null, 87, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67609, "SRR17218098", "SRX13397698", "SRS11300859", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 4", "GSM5732070", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 4", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732070", "GSM5732070: Adult Thymus 4; Danio rerio; RNA Seq", "GSM5732070", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732070", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "Fish_24_Thymus__S3_L002_I1_001.fastq.gz Fish_24_Thymus__S3_L002_R1_001.fastq.gz Fish_24_Thymus__S3_L002_R2_001.fastq.gz", "fastq fastq fastq", 18204716552.0, 144284032.0, "GSM5732070 r2", "0:8 1:27.57 2:90.60", "A:3970235370;C:2631055170;G:3048647071;T:3421683260;N:373327", 8, 27, 90, null, 3970235370, 2631055170, 3048647071, 3421683260, 373327, "SRX13397698", "SRS11300859", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.9219, null, 0.26619, null, 0.80971, null, 0.54097, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67614, "SRR17218091", "SRX13397695", "SRS11300857", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 3", "GSM5732067", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 3", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732067", "GSM5732067: Adult Thymus 3; Danio rerio; RNA Seq", "GSM5732067", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732067", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "Fish_22_Thymus__S3_L001_I1_001.fastq.gz Fish_22_Thymus__S3_L001_R1_001.fastq.gz Fish_22_Thymus__S3_L001_R2_001.fastq.gz", "fastq fastq fastq", 15755989697.0, 124869852.0, "GSM5732067 r1", "0:8 1:27.57 2:90.61", "A:3460847047;C:2243842372;G:2624369866;T:2984076004;N:733016", 8, 27, 90, null, 3460847047, 2243842372, 2624369866, 2984076004, 733016, "SRX13397695", "SRS11300857", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.91732, null, 0.27669, null, 0.81669, null, 0.55139, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [67615, "SRR17218092", "SRX13397695", "SRS11300857", "SRP350544", "PRJNA788489", "Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish", "GSE190794", "Transcriptome Analysis", "The zebrafish has proven to be a valuable model organism for studying hematopoiesis but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus elucidated B and T cell developmental trajectories and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically kidney marrows only were dissected from 3 adult GESTALT zebrafish paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation filtering and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.", null, "pubmed:35938989", null, "Adult Thymus 3", "GSM5732067", null, "tissue:sorted live thymus cells|cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "Adult Thymus 3", "The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms", "sorted live thymus cells", null, "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3\u2019 v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3\u2019 Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "Zebrafish were maintained in accordance with Boston Children\u2019s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children\u2019s Hospital ARCH guidelines.", "cell type:sorted live thymus cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in HBSS no Ca2+ or Mg2+|dissection temperature:room temperature", "GSM5732067", "GSM5732067: Adult Thymus 3; Danio rerio; RNA Seq", "GSM5732067", null, "1", "Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 \u03bcL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample assessed by Trypan blue staining and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles i7 index: 8 cycles i5 index: 0 cycles Read 2: 91 cycles. scRNA Seq", "GEO Accession:GSM5732067", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP350544", null, "loader:fastq load.py", "Fish_22_Thymus__S3_L002_I1_001.fastq.gz Fish_22_Thymus__S3_L002_R1_001.fastq.gz Fish_22_Thymus__S3_L002_R2_001.fastq.gz", "fastq fastq fastq", 15822948086.0, 125400460.0, "GSM5732067 r2", "0:8 1:27.57 2:90.61", "A:3480626521;C:2251532188;G:2629092513;T:3000199704;N:484646", 8, 27, 90, null, 3480626521, 2251532188, 2629092513, 3000199704, 484646, "SRX13397695", "SRS11300857", "SRA1342645", "GEO", "Oncology/Hematology, Boston Children's Hospital", 1, 0.91806, null, 0.27586, null, 0.81588, null, 0.5457, null, 90, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2021-12-13", "Adult", "Adult", "Thymus", "Hematopoietic System"], [76726, "SRR25288288", "SRX21031897", "SRS18304343", "SRP449631", "PRJNA994919", "Diverse Epithelial Lymphocytes in Zebrafish Revealed Using a Novel Scale Biopsy Method", "GSE237417", "Transcriptome Analysis", "Zebrafish Danio rerio are a compelling model to study lymphocytes because zebrafish and humans have similar adaptive immune systems including their lymphocytes. Antibodies that recognize zebrafish proteins are sparse so many investigators utilize transgenic lymphocyte specific fluorophore labeled lines. Human and zebrafish lymphocyte types are conserved but many aspects of zebrafish lymphocyte biology remain uninvestigated including lymphocytes in peripheral tissues like epidermis. Here we report the first study focused on zebrafish epidermal lymphocytes using scales. Obtaining zebrafish blood via non lethal methods is difficult; scales represent a source to longitudinally sample live fish. We developed a novel biopsy technique collecting scales to analyze epithelial lymphocytes from several fluorescently labeled lines. We imaged scales via confocal microscopy and demonstrated multiple lymphocyte types in scales/epidermis quantifying them flow cytometrically. We profiled gene expression of scale thymic and marrow lymphocytes from the same animals revealing B and T lineage signatures. Single cell qRT PCR and RNA sequencing scRNA seq show not only canonical B and T cells but also novel lymphocyte populations not described previously. To validate longitudinal scale biopsies we serially sampled scales from fish treated with dexamethasone DXM demonstrating epidermal lymphocyte responses. To analyze cells functionally we employed a bead ingestion assay showing thymic marrow and epidermal lymphocytes have phagocytic activity. In summary we establish a novel non lethal technique to obtain zebrafish lymphocytes providing the first quantification expression profiling and functional data DXM responses and phagocytosis from epidermal lymphocytes in the zebrafish model. Overall design: This experimental study aimed to investigate the gene expression profiles of individual lymphocytes from the zebrafish lck:GFP transgenic line by using single cell RNA sequencing scRNA seq analysis. We performed scRNA seq on GFPhi thymocytes and GFPlo scale/marrow cells from lck:GFP fish which mark different lymphocyte populations. scRNA seq data reveal diverse thymic scale and marrow lymphocyte populations for subsequent transcriptomic analysis and isolation of specific cell types.", null, "pubmed:39503619", null, "Thymus S1", "GSM7611259", null, "source name:Thymus|tissue:Thymus|cell line:NA|cell type:Lymphocytes|genotype:lck:GFP|geo loc name:missing|collection date:missing", "Thymus S1", "post conversion to fastq files reads for each sample were processed and aggregated using the 10x Genomics Cell Ranger v.6.0.0 pipeline no normalization default settings and processed in the Seurat R package v.4.3.0. We obtained transcriptomes for 6 359 cells post Cell Ranger processing. SoupX v.1.6.2 was used to model and remove ambient RNA contamination per sample and scDblFinder v.1.12.0 was used to detect potential multiplets default settings per individual tissue type. Additional QC filtering was performed to remove potential dead or dying cells along with cells exhibiting abnormal read/gene counts and high levels of mitochondrial transcripts resulting in 1 890 usable cells for our analysis. Using fastMNN cells were normalized and integrated and then clustered within Seurat Leiden algorithm. Clustering resolution was optimized using the clustree package v.0.5.0. Cluster boundaries were manually examined and fine tuned to optimize biological interpretation. Collective diagnostic gene signatures corresponding to published gene lists and our own sc qRT PCR results were explored using Seurat and UCell v.2.2.0. Preferential gene markers were determined for each distinct population using the FindAllMarkers function within Seurat to aid in cell type assignment p.adj \u2264 0.05 min.pct = 0.25. Assembly: GRCz11 Supplementary files format and content: Tab delimited value files and matrices.", "Thymus", null, "For procedures zebrafish subjects were anesthetized with 0.02% tricaine methanesulfonate. Thymus kidney marrow and scale samples were dissected and placed in 500 ul cell media RPMI + 1% FBS + 1% Pen/Strep. Single cell suspensions were prepped by dissociating tissues using a pestle and passed through 35 um filters. Various fluorescent populations were sorted from the lymphoid and precursor gates using a BD FACSJazz Instrument analysis performed in FlowJo softare. Following creation of single cell emulsions uniquely identifiable 1st strand template single cell cDNA libraries were generated from each cell by emulsion PCR. 2nd strand cDNA was generated and ligated to compatible Illumina adapters. Libraries were loaded onto single NovaSeq 6000 lanes and sequenced using read lengths of 28 bp for the first read 120 bp for the second read and 8 base index reads.", null, "tissue:Thymus|cell line:NA|cell type:Lymphocytes|genotype:lck:GFP", "GSM7611259", "GSM7611259: Thymus S1; Danio rerio; RNA Seq", "GSM7611259 r1", "GSM7611259", "1", "For procedures zebrafish subjects were anesthetized with 0.02% tricaine methanesulfonate. Thymus kidney marrow and scale samples were dissected and placed in 500 ul cell media RPMI + 1% FBS + 1% Pen/Strep. Single cell suspensions were prepped by dissociating tissues using a pestle and passed through 35 um filters. Various fluorescent populations were sorted from the lymphoid and precursor gates using a BD FACSJazz Instrument analysis performed in FlowJo softare. Following creation of single cell emulsions uniquely identifiable 1st strand template single cell cDNA libraries were generated from each cell by emulsion PCR. 2nd strand cDNA was generated and ligated to compatible Illumina adapters. Libraries were loaded onto single NovaSeq 6000 lanes and sequenced using read lengths of 28 bp for the first read 120 bp for the second read and 8 base index reads.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP449631", null, "loader:fastq load.py", "3_Mo_1ck_Thymus_S1_L001_I1_001.fastq.gz 3_Mo_1ck_Thymus_S1_L001_R1_001.fastq.gz 3_Mo_1ck_Thymus_S1_L001_R2_001.fastq.gz", "fastq fastq fastq", 13930287540.0, 89296715.0, "GSM7611259 r1", "0:8 1:28 2:120", "A:3208073091;C:2208968733;G:2525266634;T:2773161165;N:136177", 8, 28, 120, null, 3208073091, 2208968733, 2525266634, 2773161165, 136177, "SRX21031897", "SRS18304343", "SRA1673435", "Pediatrics, University of Oklahoma Health Sciences Center", "Pediatrics, University of Oklahoma Health Sciences Center", 1, 0.92048, null, 0.30227, null, 0.81708, null, 0.53081, null, 120, null, "B", null, "usable mapping rate", "illumina", "novaseq_era", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "United States", "2023-07-14", "Undetermined", "Undetermined", "Thymus", "Hematopoietic System"]], "truncated": false, "filtered_table_rows_count": 17, "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", 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