rowid,run.accession,experiment.accession,sample.accession,study.accession,bioproject,study.title,study.alias,study.type,study.abstract,study.attributes,study.PMIDs,sample.description,sample.title,sample.alias,sample.centername,sample.attributes,GEOsample.title,GEOsample.dataprocessing,GEOsample.source,GEOsample.treatmentprotocol,GEOsample.extractprotocol,GEOsample.growthprotocol,GEOsample.characteristics,GEOsample.accession,experiment.title,experiment.alias,experiment.library_name,experiment.design_description,experiment.library_construction_protocol,experiment.attributes,experiment.library_strategy,experiment.library_source,experiment.library_selection,experiment.library_layout,experiment.platform,experiment.instrument_model,experiment.spot_descriptor,experiment.study_ref,run.title,run.attributes,run.filename,run.semantic_name,run.total_bases,run.total_spots,run.alias,run.read_lengths,run.base_counts,run.r1_length,run.r2_length,run.r3_length,run.r4_length,run.Acount,run.Ccount,run.Gcount,run.Tcount,run.Ncount,run.experiment,run.pool_member,submission.accession,submission.srasource,submission.bioprojectsource,seqdetective.n_mates,seqdetective.mapping_rate.mate1,seqdetective.mapping_rate.mate2,seqdetective.nofeature_rate.mate1,seqdetective.nofeature_rate.mate2,seqdetective.sparsity.mate1,seqdetective.sparsity.mate2,seqdetective.pos_strand_rate.mate1,seqdetective.pos_strand_rate.mate2,seqdetective.readlen.mate1,seqdetective.readlen.mate2,seqdetective.judgement.mate1,seqdetective.judgement.mate2,seqdetective.judgement.reason,platform_family,instrument_generation,read_bias,selection_class,prep_kit,sc_or_bulk,tech_class,technology,tech_variant,submission.bioprojectsource.country,earliest_date,devstage_curation,devstage_curation_coarse,tissue_curation,tissue_curation_coarse
29175,SRR27292304,SRX22969963,SRS19936063,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  48 hours post burn,GSM7982886,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  48 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982886,GSM7982886: Burned Larval Zebrafish  48 hours post burn; Danio rerio; RNA Seq,GSM7982886 r1,GSM7982886,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-48hpb_S5_L001_R2_001.fastq.gz Burn-48hpb_S5_L001_R1_001.fastq.gz,fastq fastq,20874980529.0,178418637.0,GSM7982886 r1,0:28 1:89,A:5945091269;C:4392590896;G:4497688824;T:6035946283;N:3663257,28,89,,,5945091269,4392590896,4497688824,6035946283,3663257,SRX22969963,SRS19936063,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00573,0.91666,0.00222,0.19474,0.9931,0.79306,0.42082,0.53801,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29176,SRR27292305,SRX22969963,SRS19936063,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  48 hours post burn,GSM7982886,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  48 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982886,GSM7982886: Burned Larval Zebrafish  48 hours post burn; Danio rerio; RNA Seq,GSM7982886 r1,GSM7982886,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-48hpb_S5_L002_R1_001.fastq.gz Burn-48hpb_S5_L002_R2_001.fastq.gz,fastq fastq,20022973425.0,171136525.0,GSM7982886 r2,0:28 1:89,A:5709681181;C:4208386174;G:4307510275;T:5796282368;N:1113427,28,89,,,5709681181,4208386174,4307510275,5796282368,1113427,SRX22969963,SRS19936063,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00547,0.9154,0.00202,0.1955,0.99283,0.79423,0.39307,0.53427,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29177,SRR27292306,SRX22969962,SRS19936064,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  24 hours post burn,GSM7982885,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  24 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982885,GSM7982885: Burned Larval Zebrafish  24 hours post burn; Danio rerio; RNA Seq,GSM7982885 r1,GSM7982885,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-24hpb_S2_L001_R1_001.fastq.gz Burn-24hpb_S2_L001_R2_001.fastq.gz,fastq fastq,9782870058.0,83614274.0,GSM7982885 r1,0:28 1:89,A:2744737668;C:2140848416;G:2249400157;T:2645170677;N:2713140,28,89,,,2744737668,2140848416,2249400157,2645170677,2713140,SRX22969962,SRS19936064,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00752,0.92059,0.00244,0.16143,0.98944,0.82929,0.44176,0.54525,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29178,SRR27292307,SRX22969962,SRS19936064,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  24 hours post burn,GSM7982885,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  24 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982885,GSM7982885: Burned Larval Zebrafish  24 hours post burn; Danio rerio; RNA Seq,GSM7982885 r1,GSM7982885,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-24hpb_S2_L002_R2_001.fastq.gz Burn-24hpb_S2_L002_R1_001.fastq.gz,fastq fastq,9861989202.0,84290506.0,GSM7982885 r2,0:28 1:89,A:2769268861;C:2158644227;G:2263475493;T:2669311436;N:1289185,28,89,,,2769268861,2158644227,2263475493,2669311436,1289185,SRX22969962,SRS19936064,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.0076,0.9197,0.00232,0.16162,0.98995,0.82852,0.43786,0.55256,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29179,SRR27292308,SRX22969961,SRS19936062,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  6 hours post burn,GSM7982884,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  6 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982884,GSM7982884: Burned Larval Zebrafish  6 hours post burn; Danio rerio; RNA Seq,GSM7982884 r1,GSM7982884,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-6hpb_S7_L001_R1_001.fastq.gz Burn-6hpb_S7_L001_R2_001.fastq.gz,fastq fastq,12475938384.0,106631952.0,GSM7982884 r1,0:28 1:89,A:3401989108;C:2878275107;G:3114945415;T:3078525361;N:2203393,28,89,,,3401989108,2878275107,3114945415,3078525361,2203393,SRX22969961,SRS19936062,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.01053,0.89396,0.00429,0.18315,0.99458,0.85756,0.34876,0.58728,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29180,SRR27292309,SRX22969961,SRS19936062,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Burned Larval Zebrafish  6 hours post burn,GSM7982884,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn|geo loc name:missing|collection date:missing,Burned Larval Zebrafish  6 hours post burn,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Burn,GSM7982884,GSM7982884: Burned Larval Zebrafish  6 hours post burn; Danio rerio; RNA Seq,GSM7982884 r1,GSM7982884,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,Burn-6hpb_S7_L002_R1_001.fastq.gz Burn-6hpb_S7_L002_R2_001.fastq.gz,fastq fastq,11874083247.0,101487891.0,GSM7982884 r2,0:28 1:89,A:3243501055;C:2735412615;G:2958387721;T:2936117380;N:664476,28,89,,,3243501055,2735412615,2958387721,2936117380,664476,SRX22969961,SRS19936062,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.01063,0.89389,0.00439,0.18047,0.99403,0.85878,0.36393,0.54831,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29181,SRR27292310,SRX22969960,SRS19936061,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  5 dpf,GSM7982883,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  5 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982883,GSM7982883: Unwounded Larval Zebrafish  5 dpf Danio rerio; RNA Seq,GSM7982883 r1,GSM7982883,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-5dpf_S6_L001_R2_001.fastq.gz WT-5dpf_S6_L001_R1_001.fastq.gz,fastq fastq,31813158312.0,271907336.0,GSM7982883 r1,0:28 1:89,A:8991313503;C:6911976524;G:7105775004;T:8798521232;N:5572049,28,89,,,8991313503,6911976524,7105775004,8798521232,5572049,SRX22969960,SRS19936061,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00661,0.92343,0.00245,0.19293,0.99255,0.81379,0.35935,0.54818,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29182,SRR27292311,SRX22969960,SRS19936061,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  5 dpf,GSM7982883,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  5 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982883,GSM7982883: Unwounded Larval Zebrafish  5 dpf Danio rerio; RNA Seq,GSM7982883 r1,GSM7982883,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-5dpf_S6_L002_R1_001.fastq.gz WT-5dpf_S6_L002_R2_001.fastq.gz,fastq fastq,30524386113.0,260892189.0,GSM7982883 r2,0:28 1:89,A:8637605077;C:6625244702;G:6807369409;T:8452468968;N:1697957,28,89,,,8637605077,6625244702,6807369409,8452468968,1697957,SRX22969960,SRS19936061,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00648,0.92418,0.00233,0.19549,0.99241,0.81444,0.37733,0.54674,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29183,SRR27292312,SRX22969959,SRS19936060,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  4 dpf,GSM7982882,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  4 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982882,GSM7982882: Unwounded Larval Zebrafish  4 dpf Danio rerio; RNA Seq,GSM7982882 r1,GSM7982882,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-4dpf_S1_L001_R1_001.fastq.gz WT-4dpf_S1_L001_R2_001.fastq.gz,fastq fastq,12682286721.0,108395613.0,GSM7982882 r1,0:28 1:89,A:3575375325;C:2777398557;G:2900088646;T:3425909511;N:3514682,28,89,,,3575375325,2777398557,2900088646,3425909511,3514682,SRX22969959,SRS19936060,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00505,0.92112,0.00185,0.16865,0.99257,0.8258,0.38216,0.5512,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29184,SRR27292313,SRX22969959,SRS19936060,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  4 dpf,GSM7982882,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  4 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982882,GSM7982882: Unwounded Larval Zebrafish  4 dpf Danio rerio; RNA Seq,GSM7982882 r1,GSM7982882,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-4dpf_S1_L002_R2_001.fastq.gz WT-4dpf_S1_L002_R1_001.fastq.gz,fastq fastq,12832453413.0,109679089.0,GSM7982882 r2,0:28 1:89,A:3620789787;C:2810897812;G:2929508681;T:3469577031;N:1680102,28,89,,,3620789787,2810897812,2929508681,3469577031,1680102,SRX22969959,SRS19936060,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.0052,0.92355,0.00192,0.16775,0.99263,0.8253,0.37089,0.55115,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29185,SRR27292314,SRX22969958,SRS19936059,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  3 dpf,GSM7982881,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  3 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982881,GSM7982881: Unwounded Larval Zebrafish  3 dpf Danio rerio; RNA Seq,GSM7982881 r1,GSM7982881,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-3dpf_S4_L001_R1_001.fastq.gz WT-3dpf_S4_L001_R2_001.fastq.gz,fastq fastq,7586138430.0,64838790.0,GSM7982881 r1,0:28 1:89,A:2101749534;C:1692875352;G:1784872358;T:2005308370;N:1332816,28,89,,,2101749534,1692875352,1784872358,2005308370,1332816,SRX22969958,SRS19936059,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00976,0.92096,0.00368,0.18077,0.99356,0.83875,0.3661,0.59073,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
29186,SRR27292315,SRX22969958,SRS19936059,SRP479046,PRJNA1054617,Modeling Neutrophil Heterogeneity Contribution to Burn Healing in Larval Zebrafish,GSE250610,Transcriptome Analysis,Neutrophils accumulate early in burn wounds  and their activation is associated with more severe burns. Understanding their functionality would facilitate the development of a more targeted therapeutic strategy for healing. However  we still lack a view of the cellular and functional heterogeneity in neutrophils involved in thermal injuries. Here  we establish the use of larval zebrafish for understanding neutrophil responses in burn. Zebrafish model allows for linking neutrophil states and their functions in real time through genetic modifications and live imaging. We performed single cell transcriptional scRNA Seq mapping of myeloid cells during a 3 day time course in burn and unwounded conditions. We identified transcriptionally distinct states in myeloid cells that form a consistent population structure across time points and conditions. By comparing burn and unwounded conditions  we found subtype specific enrichment of biological processes and differential usage of gene regulatory networks. Pseudotime and RNA velocity analyses predict distinct branched trajectories for neutrophils  with one branch resembling the process of human neutrophil maturation. The other branch is not transcriptionally conserved with humans and is highly associated with leukocyte migration functionality  suggesting its engagement at the wound site. Transcriptional network analysis identified RAR/RXR family transcription factors as potential upstream factors driving this trajectory divergence in neutrophils. Furthermore  we characterized the transcriptional dynamics of cell cell interactions in both conditions by time. Among burn induced signaling pathways  we found il6 il6r signaling as a time point specific macrophage neutrophil interaction  suggesting the importance of timing in innate immune response in burn. Finally  to test the translational value of our fish model  we examined zebrafish neutrophil state signatures in human burn patient samples. We found homolog expression of immature neutrophils positively correlates with the degree of total body surface area TBSA in patients. Flow cytometry confirmed the presence of neutrophils carrying these signatures in patient's blood. Our findings demonstrate the potential of using zebrafish as a model to identify early innate immune response signatures that could inform a timely treatment of burn in humans. This work builds the molecular foundation and a comparative single cell genomic framework to guide future identification of actionable pathways to burn wound healing in patients. Overall design: At 6/24/48 hour post burn hpb  burned and unwounded larvae at matching developmental stages 3/4/5 dpf dpf were transferred to 35 mm dish containing calcium free PBS for 15 min and then anesthetized with tricaine methanesulfonate MS222  200 mg/L. A total of 150 fish were used for each time point by condition. Each dish of fish was digested with 2 mL digestion solution 0.25% trypsin  1mM EDTA in PBS at 28.5°C for 90 min with gentle pipetting every 10 min. Digestion was stopped by adding 200 mL digestion stop solution 1mM CaCl2  100% FBS. Dissociated cells were filtered with 40 mm cell strainers and centrifuged for 3 min at 3 000 rpm at 4 °C. Cell pellets were resuspended in PBS with 10% FBS plus DAPI 1mg/mL. Fluorescence activated cell sorting FACS was performed at University of Wisconsin Carbone Cancer Center Flow Lab using BD FACSAria. Targeted cells passed gating for cells versus debris  singles versus doublets  live versus dead and were high in either 488 nm/561 nm channel. Both dendra+ 488 nm channel and mCherry+ 561 nm channel cells were sorted into PBS with 10% FBS. Post sorting  cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin Madison Biotechnology Center RRID: SCR 017757. Samples were processed with Chromium Single Cell Gene Expression Solution three prime v2 10X Genomics.,,pubmed:38922186,,Unwounded Larval Zebrafish  3 dpf,GSM7982881,,source name:Whole blood|tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded|geo loc name:missing|collection date:missing,Unwounded Larval Zebrafish  3 dpf,FASTQ files were aligned to Zebrafish genome using cellranger version v3.1.1 Unwounded 4days  Burn 24 hours  v6.1.2 Unwounded 3days  Unwounded 5days  Burn 6hours  Burn 48hours UMI cutoffs per droplet were calcuated from cellranger and filtered feature matrices were used to generate a Seurat object V4 Assembly: GRCz11 danRer11 Supplementary files format and content: RNA count matrix,Whole blood,Larval zebrafish were wounded on the tail fin region at 3 days post feritlization using a handheld cauterizer.,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,Zebrafish embroys were collected from adult Tgmpx:dendra mpeg1.1:mCherry fish and kept in E3 medium with 1% methylene blue inside a 28.5˚C incubator.,tissue:Whole blood|cell type:Macrophage|cell type:Neutrophil|genotype:WT|treatment:Unwounded,GSM7982881,GSM7982881: Unwounded Larval Zebrafish  3 dpf Danio rerio; RNA Seq,GSM7982881 r1,GSM7982881,1,Dissociated cells were sorted using fluorescence activated cell sorting using BD FACSAria to separate neutrophils and macrophages. Cells were then loaded into the 10x chromium controller for GEM encapsulation and reverse transcription per manufacturer's instructions. Library construction was performed per 10x instructions with three prime v2 chemistry Each prepared library was sequenced on the NovaSeq using dual index sequencing,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP479046,,loader:fastq load.py,WT-3dpf_S4_L002_R1_001.fastq.gz WT-3dpf_S4_L002_R2_001.fastq.gz,fastq fastq,7258692168.0,62040104.0,GSM7982881 r2,0:28 1:89,A:2013529524;C:1617959197;G:1704796898;T:1922004726;N:401823,28,89,,,2013529524,1617959197,1704796898,1922004726,401823,SRX22969958,SRS19936059,SRA1772557,University of Wisconsin-Madison,University of Wisconsin-Madison,2,0.00949,0.92207,0.00359,0.18081,0.99375,0.83751,0.37347,0.55889,28,89,T,B,sc-like readlen,illumina,novaseq_era,unknown,random_priming,unknown,sc,single_cell_droplet,10x,,United States,2023-12-19,Multi-stage,Multi-stage,Blood,Hematopoietic System
35500,SRR32834046,SRX28117231,SRS24473011,SRP572644,PRJNA1241156,Single Cell Atlas Reveals Abundant Innate Lymphoid Cells in Larval Zebrafish,GSE292726,Transcriptome Analysis,Innate Lymphoid cells ILCs are a group of tissue resident lymphocytes that participate in immune defense and tissue homeostasis. The existence and ontogeny of ILCs during zebrafish embryonic development are not defined. During embryogenesis  hematopoietic stem cells HSCs and HSC independent Progenitor Cells HPCs form contemporaneously and produce overlapping and distinct immune cell types. To delineate when ILCs emerge and if they originate exclusively from HSCs or HPCs  we performed lineage tracing of the emerging hematopoietic system during larval development followed by single cell RNA sequencing of HSC and HPC progeny. To determine if the ILC like cells are derived from the definitive hematopoietic wave  we did lineage tracing and scRNA seq experiment in runx1W84X mutants and illustrated that all ILC like clusters are Runx1 dependent. We confirmed that the transcriptionally detected larval ILC like cells are Rag independent and Il2rg dependent. Lastly  embryonic ILCs are functional because they respond to an immune system stimulation with a viral mimic. The work provides fundamental knowledge on the early establishment of immune hierarchies and opens the zebrafish model to the exploration of ILC origination and function. Overall design: Experiment 1: for lineage tracing experiment  HSPCs were labled with mCherry at 3 timepoints in development: either at 1  2  or 3 dpf. The differentiated progeny of labled HSPCs were sorted out from whole zebrafish using FACS at one day post labelling  6 dpf  and 10 dpf. Experiment 2: for Runx1 mutants experiment  early HSPCs 1dpf derived mCherry cells were isolated from sibling controls or homozygous runx1W84X mutants at 6 dpf from whole zebrafish. Experiment 3: for rag1/R848 experiment  mCherry cells derived from early HSPCs 1dpf were isolated at 8 dpf from slc24a5 crispant controls  rag1 crispants and from slc24a5 and rag1 crispants treated with R848 agonist.,,,,mCherry cells  early HSPCs 2 dpf,GSM8864259,,tissue:Hematopoietic & Immune Cells|cell type:Hematopoietic & Immune Cells|geo loc name:missing|collection date:missing,mCherry cells  early HSPCs 2 dpf,For experiments 1 lineage tracing and 2 runx1 mutants  CellRanger 7.0.1 was used. For experiment 3 slc24a5/rag1/R848  CellRanger 8.0.1 was used. The standard count function in Cell Ranger was used for all samples. Assembly: Danie rerio GRCz11 Supplementary files format and content: Matrix files CellRanger output filtered feature bc matrix,Hematopoietic & Immune Cells,The slc24a5 and rag1 crispants were treated with 25uM of Resiquimod R848 at 6 dpf,Zebrafish larvae were minced with a razor blade and collected into PBS. The suspension was then digested with Liberase for 15 min at 37C. FBS was used to inactivate the librase. Cells were filtered once before isolating via FACS. Libraries were prepared according to manufacturer's instructions of the kits. For experiments 1 lineage tracing & 2 runx1 mutants  the Chromium Next GEM Single Cell three prime Kit v3.1 was used. For experiment 3 slc24a5/rag1/R848  the Chromium Next GEM Single Cell three prime Kit v4 was used.,Larvae were maintained in standard conditions  at 28C,cell type:Hematopoietic & Immune Cells,GSM8864259,GSM8864259: mCherry cells  early HSPCs 2 dpf; Danio rerio; RNA Seq,GSM8864259 r1,GSM8864259,1,Zebrafish larvae were minced with a razor blade and collected into PBS. The suspension was then digested with Liberase for 15 min at 37C. FBS was used to inactivate the librase. Cells were filtered once before isolating via FACS. Libraries were prepared according to manufacturer's instructions of the kits. For experiments 1 lineage tracing & 2 runx1 mutants  the Chromium Next GEM Single Cell three prime Kit v3.1 was used. For experiment 3 slc24a5/rag1/R848  the Chromium Next GEM Single Cell three prime Kit v4 was used.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP572644,,loader:fastq load.py|options:  readTypes=TBTT   read1PairFiles=earlyHSPC 2dpf S5 L003 R1 001.fastq.gz   read2PairFiles=earlyHSPC 2dpf S5 L003 R2 001.fastq.gz   read3PairFiles=earlyHSPC 2dpf S5 L003 I1 001.fastq.gz   read4PairFiles=earlyHSPC 2dpf S5 L003 I2 001.fastq.gz,earlyHSPC_2dpf_S5_L003_I1_001.fastq.gz earlyHSPC_2dpf_S5_L003_I2_001.fastq.gz earlyHSPC_2dpf_S5_L003_R1_001.fastq.gz earlyHSPC_2dpf_S5_L003_R2_001.fastq.gz,fastq fastq fastq fastq,80730897280.0,252284054.0,GSM8864259 r1,0:150 1:150 2:10 3:10,A:10564796609;C:7860706889;G:9394625294;T:8808848403;N:1213630905,150,150,10,10,10564796609,7860706889,9394625294,8808848403,1213630905,SRX28117231,SRS24473011,SRA2098862,Albert Einstein College of Medicine,Albert Einstein College of Medicine,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2025-03-24,Multi-stage,Multi-stage,Blood,Hematopoietic System
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.,,pubmed:35938989,,Juvenile  Thymus  4,GSM5732082,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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:GSM5732082,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,4wpfThymus_6_S4_L001_I1_001.fastq.gz 4wpfThymus_6_S4_L001_R1_001.fastq.gz 4wpfThymus_6_S4_L001_R2_001.fastq.gz,fastq fastq fastq,14767492309.0,116279467.0,GSM5732082 r1,0:8 1:28 2:91,A:3176407665;C:2147673056;G:2551453338;T:2705740495;N:156943,8,28,91,,3176407665,2147673056,2551453338,2705740495,156943,SRX13397710,SRS11300870,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.87149,,0.22037,,0.84049,,0.53423,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Multi-stage,Multi-stage,Thymus,Hematopoietic System
67585,SRR17218122,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.,,pubmed:35938989,,Juvenile  Thymus  4,GSM5732082,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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:GSM5732082,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,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,,3154820274,2128697452,2532561232,2688755742,121853,SRX13397710,SRS11300870,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.87166,,0.22046,,0.8407,,0.5334,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  3,GSM5732081,,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. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted live thymus cells,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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:GSM5732081,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,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,,3107408243,2119850736,2503503910,2650750983,154425,SRX13397709,SRS11300871,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.85944,,0.20997,,0.84064,,0.53634,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  3,GSM5732081,,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. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted live thymus cells,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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:GSM5732081,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,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,,3079316058,2096769810,2479668921,2628218918,118657,SRX13397709,SRS11300871,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.85981,,0.21085,,0.8393,,0.52667,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  2,GSM5732080,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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,,SRP350544,,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,,3740652508,2562291967,3023715848,3183446304,184166,SRX13397708,SRS11300869,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.84416,,0.21191,,0.83897,,0.53108,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  2,GSM5732080,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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,,SRP350544,,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,,3695117374,2526230853,2985988000,3146489343,141692,SRX13397708,SRS11300869,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.84569,,0.21301,,0.83899,,0.53404,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  1,GSM5732079,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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,,SRP350544,,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,,3503038095,2359078159,2792714907,2968200161,171269,SRX13397707,SRS11300868,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.84501,,0.21705,,0.8379,,0.53566,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,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.,,pubmed:35938989,,Juvenile  Thymus  1,GSM5732079,,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,,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 μL 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’ 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’ 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’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s 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,,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 μL 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,,SRP350544,,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,,3472769520,2334135014,2767323491,2943866297,131761,SRX13397707,SRS11300868,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.84624,,0.21812,,0.83918,,0.52823,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Multi-stage,Multi-stage,Thymus,Hematopoietic System
76523,SRR24999167,SRX20755040,SRS18043617,SRP445421,PRJNA986547,Znf687 recruits Brd4 Smrt complex  to regulate gfi1aa expression   during neutrophil development,GSE235609,Transcriptome Analysis,Neutrophils are key component of the innate immune system in vertebrates. Diverse transcription factors and cofactors act in a well coordinated manner to ensure proper neutrophil development. Dysregulation of the transcriptional program triggering neutrophil differentiation is associated with various human hematologic disorders such as neutropenia  neutrophilia  and leukemia. In the current study we show the zinc finger protein Znf687 is a lineage specific transcription factor  whose deficiency leads to an impaired neutrophil development in zebrafish. Mechanistically  Znf687 functions as a negative regulator of gfi1aa  a pivotal modulator in terminal granulopoiesis  to regulate neutrophil maturation. Moreover  we found BRD4  an important epigenetic regulator  interacts with ZNF687 in neutrophils. Deficiency of brd4 results in similar defective neutrophil development as observed in znf687 mutant zebrafish. Biochemical and genetic analyses further reveal that instead of serving as a canonical transcriptional coactivator  Brd4 directly interacts and bridges Znf687 and Smrt nuclear corepressor on gfi1aa gene's promoter to exert transcription repression. Overall  our work not only indicates Znf687 and Brd4 are reciprocally required in promoting granulopoiesis  but also provides new insights into the role of the two crucial regulators in transcriptional repression. Overall design: We found that Znf687 served as a transcription repressor.we speculated that the aberrant upregulation of certain downstream target of Znf687 would be responsible for the defective neutrophil differentiation. Then RNA sequencing RNA seq analyses were conducted in the remaining mpx+ cells isolated from Tgmpx:eGFP and znf687a MO injected Tgmpx:eGFP larvae at 48 hpf.,,pubmed:38326409,,Tgmpx:eGFP larvae injected with znf687a MO 1,GSM7506192,,source name:hematopoietic|tissue:hematopoietic|cell line:mpx+|cell type:neutrophil|genotype:znf687a knowdown|treatment:znf687a MO|geo loc name:missing|collection date:missing,Tgmpx:eGFP larvae injected with znf687a MO 1,We filter the low quality reads More than 20% of the bases qualities are lower than 10  reads with adaptors and reads with unknown bases N bases more than 5% to get the clean reads. We use fastp to filter. We use HISATv2.0.4 to do the mapping step. We mapped clean reads to reference using Bowtie2v2.2.6   and then calculate gene expression level with RSEMv1.2.12. Then  we calculate pearson correlation between all samples using cor  perform hierarchical clustering between all samples using hclust  perform PCA analysis with all samples using princomp  and draw the diagrams with ggplot2 with fuctions of R. Assembly: GRCz11 Supplementary files format and content: tab delimited text files include RPKM values for each Sample,hematopoietic,mpx+ cells isolated from Tgmpx:eGFP and znf687a MO injected Tgmpx:eGFP larvae at 48 hpf.,RNA was extracted from sorted cells using RNeasy Micro Qiagen  Manchester  UK 1 μg total RNA was used for following library preparation. Paired end libraries were prepared using the VAHTS® Universal V8 RNA seq Library Prep Kit vazyme Technology Co.  Ltd.  Nanjing  China .The polyA mRNA isolation was performed using OligodT beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second strand cDNA were synthesized. The purified double stranded cDNA was then treated to repair both ends and add a dA tailing in one reaction  followed by a T A ligation to add adaptors to both ends. Size selection of Adaptor ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Navoseq6000 instrument for sequencing using a 2x150 paired end PE configuration according to manufacturer’s instructions.,Wild type zebrafish without xxx were used as blank to determine the background values in GFP controls.,tissue:hematopoietic|cell line:mpx+|cell type:neutrophil|genotype:znf687a knowdown|treatment:znf687a  MO,GSM7506192,GSM7506192: Tgmpx:eGFP larvae injected with znf687a MO 1; Danio rerio; RNA Seq,GSM7506192 r1,GSM7506192,1,RNA was extracted from sorted cells using RNeasy Micro Qiagen  Manchester  UK 1 μg total RNA was used for following library preparation. Paired end libraries were prepared using the VAHTS® Universal V8 RNA seq Library Prep Kit vazyme Technology Co.  Ltd.  Nanjing  China .The polyA mRNA isolation was performed using OligodT beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second strand cDNA were synthesized. The purified double stranded cDNA was then treated to repair both ends and add a dA tailing in one reaction  followed by a T A ligation to add adaptors to both ends. Size selection of Adaptor ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Navoseq6000 instrument for sequencing using a 2x150 paired end PE configuration according to manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP445421,,,R21136162-qtsw-z003-mo-11-10_combined_R2.fastq.gz R21136162-qtsw-z003-mo-11-10_combined_R1.fastq.gz,fastq fastq,5474240400.0,18247468.0,GSM7506192 r1,0:150 1:150,A:1344187663;C:1136523926;G:1697034586;T:1296453731;N:40494,150,150,,,1344187663,1136523926,1697034586,1296453731,40494,SRX20755040,SRS18043617,SRA1660768,"CNRS-LIA Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics","CNRS-LIA Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics",2,0.85041,0.8461,0.08081,0.07986,0.74328,0.746,0.48356,0.47751,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,China,2023-06-22,Multi-stage,Multi-stage,Blood,Hematopoietic System
76524,SRR24999168,SRX20755039,SRS18043616,SRP445421,PRJNA986547,Znf687 recruits Brd4 Smrt complex  to regulate gfi1aa expression   during neutrophil development,GSE235609,Transcriptome Analysis,Neutrophils are key component of the innate immune system in vertebrates. Diverse transcription factors and cofactors act in a well coordinated manner to ensure proper neutrophil development. Dysregulation of the transcriptional program triggering neutrophil differentiation is associated with various human hematologic disorders such as neutropenia  neutrophilia  and leukemia. In the current study we show the zinc finger protein Znf687 is a lineage specific transcription factor  whose deficiency leads to an impaired neutrophil development in zebrafish. Mechanistically  Znf687 functions as a negative regulator of gfi1aa  a pivotal modulator in terminal granulopoiesis  to regulate neutrophil maturation. Moreover  we found BRD4  an important epigenetic regulator  interacts with ZNF687 in neutrophils. Deficiency of brd4 results in similar defective neutrophil development as observed in znf687 mutant zebrafish. Biochemical and genetic analyses further reveal that instead of serving as a canonical transcriptional coactivator  Brd4 directly interacts and bridges Znf687 and Smrt nuclear corepressor on gfi1aa gene's promoter to exert transcription repression. Overall  our work not only indicates Znf687 and Brd4 are reciprocally required in promoting granulopoiesis  but also provides new insights into the role of the two crucial regulators in transcriptional repression. Overall design: We found that Znf687 served as a transcription repressor.we speculated that the aberrant upregulation of certain downstream target of Znf687 would be responsible for the defective neutrophil differentiation. Then RNA sequencing RNA seq analyses were conducted in the remaining mpx+ cells isolated from Tgmpx:eGFP and znf687a MO injected Tgmpx:eGFP larvae at 48 hpf.,,pubmed:38326409,,Tgmpx:eGFP larvae injected with znf687a MO 2,GSM7506193,,source name:hematopoietic|tissue:hematopoietic|cell line:mpx+|cell type:neutrophil|genotype:znf687a knowdown|treatment:znf7687a MO|geo loc name:missing|collection date:missing,Tgmpx:eGFP larvae injected with znf687a MO 2,We filter the low quality reads More than 20% of the bases qualities are lower than 10  reads with adaptors and reads with unknown bases N bases more than 5% to get the clean reads. We use fastp to filter. We use HISATv2.0.4 to do the mapping step. We mapped clean reads to reference using Bowtie2v2.2.6   and then calculate gene expression level with RSEMv1.2.12. Then  we calculate pearson correlation between all samples using cor  perform hierarchical clustering between all samples using hclust  perform PCA analysis with all samples using princomp  and draw the diagrams with ggplot2 with fuctions of R. Assembly: GRCz11 Supplementary files format and content: tab delimited text files include RPKM values for each Sample,hematopoietic,mpx+ cells isolated from Tgmpx:eGFP and znf687a MO injected Tgmpx:eGFP larvae at 48 hpf.,RNA was extracted from sorted cells using RNeasy Micro Qiagen  Manchester  UK 1 μg total RNA was used for following library preparation. Paired end libraries were prepared using the VAHTS® Universal V8 RNA seq Library Prep Kit vazyme Technology Co.  Ltd.  Nanjing  China .The polyA mRNA isolation was performed using OligodT beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second strand cDNA were synthesized. The purified double stranded cDNA was then treated to repair both ends and add a dA tailing in one reaction  followed by a T A ligation to add adaptors to both ends. Size selection of Adaptor ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Navoseq6000 instrument for sequencing using a 2x150 paired end PE configuration according to manufacturer’s instructions.,Wild type zebrafish without xxx were used as blank to determine the background values in GFP controls.,tissue:hematopoietic|cell line:mpx+|cell type:neutrophil|genotype:znf687a knowdown|treatment:znf7687a MO,GSM7506193,GSM7506193: Tgmpx:eGFP larvae injected with znf687a MO 2; Danio rerio; RNA Seq,GSM7506193 r1,GSM7506193,1,RNA was extracted from sorted cells using RNeasy Micro Qiagen  Manchester  UK 1 μg total RNA was used for following library preparation. Paired end libraries were prepared using the VAHTS® Universal V8 RNA seq Library Prep Kit vazyme Technology Co.  Ltd.  Nanjing  China .The polyA mRNA isolation was performed using OligodT beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second strand cDNA were synthesized. The purified double stranded cDNA was then treated to repair both ends and add a dA tailing in one reaction  followed by a T A ligation to add adaptors to both ends. Size selection of Adaptor ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Navoseq6000 instrument for sequencing using a 2x150 paired end PE configuration according to manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP445421,,,R21136162-qtsw-z003-mo-11-11_combined_R2.fastq.gz R21136162-qtsw-z003-mo-11-11_combined_R1.fastq.gz,fastq fastq,7189581900.0,23965273.0,GSM7506193 r1,0:150 1:150,A:1383671914;C:1160994305;G:3299098586;T:1345763504;N:53591,150,150,,,1383671914,1160994305,3299098586,1345763504,53591,SRX20755039,SRS18043616,SRA1660768,"CNRS-LIA Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics","CNRS-LIA Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics",2,0.89402,0.8908,0.09186,0.09076,0.76307,0.76473,0.51884,0.5204,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,China,2023-06-22,Multi-stage,Multi-stage,Blood,Hematopoietic System