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 10060,ERR4795364,ERX4665135,ERS5281176,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 4,E MTAB 9727:Sample 4,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 4 s,Sample 4 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 4,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN4_AH2TW3BGX5_S1_R1_cat.fastq.gz,fastq,5410723202.0,71707309.0,E MTAB 9727:Sample 4,0:75.46 1:0,A:1330416803;C:531269504;G:734031980;T:2814959249;N:45666,75,0,,,1330416803,531269504,734031980,2814959249,45666,ERX4665135,ERS5281176,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.33614,,0.21532,,0.99019,,0.41002,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10061,ERR4795365,ERX4665135,ERS5281176,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 4,E MTAB 9727:Sample 4,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 4 s,Sample 4 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 4,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN4_AH2TW3BGX5_S1_R2_cat.fastq.gz,fastq,5414184547.0,71707309.0,E MTAB 9727:Sample 4 1,0:0 1:75.50,A:1582938104;C:1052565419;G:1177180395;T:1600161662;N:1338967,0,75,,,1582938104,1052565419,1177180395,1600161662,1338967,ERX4665135,ERS5281176,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.84976,,0.28521,,0.85038,,0.5124,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10062,ERR4795362,ERX4665134,ERS5281175,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 3,E MTAB 9727:Sample 3,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 3 s,Sample 3 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 3,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN3_AHY3WGBGX3_S7_R1_cat.fastq.gz,fastq,4823193891.0,63965519.0,E MTAB 9727:Sample 3,0:75.40 1:0,A:1323978162;C:446831209;G:581746343;T:2470114091;N:524086,75,0,,,1323978162,446831209,581746343,2470114091,524086,ERX4665134,ERS5281175,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.36549,,0.19722,,0.95552,,0.4702,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10063,ERR4795363,ERX4665134,ERS5281175,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 3,E MTAB 9727:Sample 3,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 3 s,Sample 3 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 3,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN3_AHY3WGBGX3_S7_R2_cat.fastq.gz,fastq,4828889391.0,63965519.0,E MTAB 9727:Sample 3 1,0:0 1:75.49,A:1434099697;C:964508763;G:893584575;T:1534727600;N:1968756,0,75,,,1434099697,964508763,893584575,1534727600,1968756,ERX4665134,ERS5281175,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.85811,,0.26231,,0.82731,,0.48618,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10064,ERR4795360,ERX4665133,ERS5281174,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 2,E MTAB 9727:Sample 2,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 2 s,Sample 2 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 2,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN2_AHY3WGBGX3_S6_R1_cat.fastq.gz,fastq,5598371393.0,74235388.0,E MTAB 9727:Sample 2,0:75.41 1:0,A:1526486973;C:558020193;G:717587491;T:2795646081;N:630655,75,0,,,1526486973,558020193,717587491,2795646081,630655,ERX4665133,ERS5281174,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.28059,,0.19252,,0.96404,,0.4874,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10065,ERR4795361,ERX4665133,ERS5281174,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 2,E MTAB 9727:Sample 2,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 2 s,Sample 2 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 2,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN2_AHY3WGBGX3_S6_R2_cat.fastq.gz,fastq,5600151371.0,74235388.0,E MTAB 9727:Sample 2 1,0:0 1:75.44,A:1850568176;C:1035622971;G:1071138281;T:1640475883;N:2346060,0,75,,,1850568176,1035622971,1071138281,1640475883,2346060,ERX4665133,ERS5281174,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.7679,,0.30371,,0.82651,,0.43401,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10066,ERR4795358,ERX4665132,ERS5281173,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 1,E MTAB 9727:Sample 1,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 1 s,Sample 1 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 1,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN1_AHY3WGBGX3_S5_R1_cat.fastq.gz,fastq,4996502316.0,66258508.0,E MTAB 9727:Sample 1,0:75.41 1:0,A:1330744174;C:451622042;G:591151500;T:2622422664;N:561936,75,0,,,1330744174,451622042,591151500,2622422664,561936,ERX4665132,ERS5281173,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.29754,,0.17671,,0.9669,,0.45463,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 10067,ERR4795359,ERX4665132,ERS5281173,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration and their dysregulation can result in congenital anomalies of the viscera ventral wall defects and mesothelioma tumors. Nonetheless the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here we combine genetic lineage tracing in toto live imaging and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos we chart the origin of mesothelial progenitors to the lateral most hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium we find de novo expression of LPM associated transcription factors and in particular of Hand2 indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2 contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Sample 1,E MTAB 9727:Sample 1,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 1 s,Sample 1 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3 without xxx glutamine and sodium pyruvate Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing the cells were resuspended in 1X PBS. drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al. 2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch a 4 bp random molecular barcode UMI a cell specific barcode the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter provided by Single Cell Discoveries. post sorting the plates were immediately placed on ice and stored at −80°C. In brief ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al. 2016. To generate sequencing libraries RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 1,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN1_AHY3WGBGX3_S5_R2_cat.fastq.gz,fastq,4999908389.0,66258508.0,E MTAB 9727:Sample 1 1,0:0 1:75.46,A:1591945117;C:933968124;G:986108614;T:1485824022;N:2062512,0,75,,,1591945117,933968124,986108614,1485824022,2062512,ERX4665132,ERS5281173,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.81333,,0.2466,,0.83027,,0.51572,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 25228,SRR25685572,SRX21410761,SRS18649243,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 14dpi heart3,GSM7717530,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi|geo loc name:missing|collection date:missing,zebrafish 14dpi heart3,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi,GSM7717530,GSM7717530: zebrafish 14dpi heart3; Danio rerio; RNA Seq,GSM7717530 r1,GSM7717530,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,DB14zf4_R1.fq DB14zf4_R2.fq,fastq fastq,571157496.0,7312520.0,GSM7717530 r1,,A:132329616;C:53169305;G:72431948;T:313058610;N:168017,,,,,132329616,53169305,72431948,313058610,168017,SRX21410761,SRS18649243,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.01414,0.679,0.01413,0.08289,1.0,0.98395,,0.48915,76,76,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25229,SRR25685573,SRX21410760,SRS18649241,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 14dpi heart2,GSM7717529,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi|geo loc name:missing|collection date:missing,zebrafish 14dpi heart2,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi,GSM7717529,GSM7717529: zebrafish 14dpi heart2; Danio rerio; RNA Seq,GSM7717529 r1,GSM7717529,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,DB14zf3_R1.fq DB14zf3_R2.fq,fastq fastq,97890367.0,1292037.0,GSM7717529 r1,,A:21637243;C:8563955;G:12004508;T:55662880;N:21781,,,,,21637243,8563955,12004508,55662880,21781,SRX21410760,SRS18649241,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.0,0.689,0.0,0.07655,1.0,0.99831,,0.63709,76,75,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25230,SRR25685574,SRX21410759,SRS18649242,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 14dpi heart1,GSM7717528,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi|geo loc name:missing|collection date:missing,zebrafish 14dpi heart1,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:14dpi,GSM7717528,GSM7717528: zebrafish 14dpi heart1; Danio rerio; RNA Seq,GSM7717528 r1,GSM7717528,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,DB14zf1_R1.fq DB14zf1_R2.fq,fastq fastq,1031757474.0,7257847.0,GSM7717528 r1,,A:276216587;C:146620316;G:184052670;T:423474577;N:1393324,,,,,276216587,146620316,184052670,423474577,1393324,SRX21410759,SRS18649242,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.04176,0.6198,0.03023,0.08234,0.99894,0.88885,0.64556,0.53126,76,75,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25231,SRR25685575,SRX21410758,SRS18649240,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 7dpi heart3,GSM7717527,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi|geo loc name:missing|collection date:missing,zebrafish 7dpi heart3,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi,GSM7717527,GSM7717527: zebrafish 7dpi heart3; Danio rerio; RNA Seq,GSM7717527 r1,GSM7717527,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,D37DPIZF3_R1.fq D37DPIZF3_R2.fq,fastq fastq,898901460.0,7585924.0,GSM7717527 r1,,A:236935836;C:100995680;G:128458892;T:431620821;N:890231,,,,,236935836,100995680,128458892,431620821,890231,SRX21410758,SRS18649240,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.12082,0.70068,0.10594,0.1234,0.99983,0.90136,0.875,0.48346,75,76,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25232,SRR25685576,SRX21410757,SRS18649239,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 7dpi heart2,GSM7717526,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi|geo loc name:missing|collection date:missing,zebrafish 7dpi heart2,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi,GSM7717526,GSM7717526: zebrafish 7dpi heart2; Danio rerio; RNA Seq,GSM7717526 r1,GSM7717526,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,D27DPIZF2_R1.fq D27DPIZF2_R2.fq,fastq fastq,1060429257.0,7609110.0,GSM7717526 r1,,A:283107752;C:130181128;G:159305328;T:486464477;N:1370572,,,,,283107752,130181128,159305328,486464477,1370572,SRX21410757,SRS18649239,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.19472,0.68783,0.17584,0.16739,0.99967,0.85628,0.44,0.56319,75,76,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25233,SRR25685577,SRX21410756,SRS18649238,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 7dpi heart1,GSM7717525,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi|geo loc name:missing|collection date:missing,zebrafish 7dpi heart1,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:7dpi,GSM7717525,GSM7717525: zebrafish 7dpi heart1; Danio rerio; RNA Seq,GSM7717525 r1,GSM7717525,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,loader:fastq load.py|options: allowEarlyFileEnd,D17DPIZF1_R1.fq D17DPIZF1_R2.fq,fastq fastq,1073044507.0,7624815.0,GSM7717525 r1,,A:288576038;C:136858647;G:170330711;T:475869519;N:1409592,,,,,288576038,136858647,170330711,475869519,1409592,SRX21410756,SRS18649238,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.15938,0.70435,0.14583,0.08079,0.99981,0.87949,0.38461,0.57027,75,76,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25234,SRR25685578,SRX21410755,SRS18649237,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 3dpi heart3,GSM7717524,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi|geo loc name:missing|collection date:missing,zebrafish 3dpi heart3,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi,GSM7717524,GSM7717524: zebrafish 3dpi heart3; Danio rerio; RNA Seq,GSM7717524 r1,GSM7717524,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,,FKHEART3_R1.fastq FKHEART3_R2.fastq,fastq fastq,781079621.0,5184230.0,GSM7717524 r1,0:75.26 1:75.40,A:277491111;C:118539142;G:116911958;T:268048500;N:88910,75,75,,,277491111,118539142,116911958,268048500,88910,SRX21410755,SRS18649237,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.04348,0.71869,0.04347,0.28823,1.0,0.79681,,0.53909,76,75,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25235,SRR25685579,SRX21410754,SRS18649236,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 3dpi heart2,GSM7717523,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi|geo loc name:missing|collection date:missing,zebrafish 3dpi heart2,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi,GSM7717523,GSM7717523: zebrafish 3dpi heart2; Danio rerio; RNA Seq,GSM7717523 r1,GSM7717523,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,,FKHEART2_R1.fastq FKHEART2_R2.fastq,fastq fastq,684820059.0,4547520.0,GSM7717523 r1,0:75.21 1:75.38,A:258487418;C:93776953;G:97187917;T:235293267;N:74504,75,75,,,258487418,93776953,97187917,235293267,74504,SRX21410754,SRS18649236,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.09406,0.79542,0.09405,0.0802,1.0,0.83029,,0.56367,75,75,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25236,SRR25685580,SRX21410753,SRS18649235,SRP455780,PRJNA1006660,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [TOMOseq mouse and zebrafish],GSE241156,Other,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,zebrafish 3dpi heart1,GSM7717522,,source name:adult heart|tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi|geo loc name:missing|collection date:missing,zebrafish 3dpi heart1,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 processed data files contain transcript read counts normalized against spike in read count and total read count per section Assembly: GRCm38 for mouse danrer9 for zebrafish Supplementary files format and content: csv file containing transcript counts per gene rows and section columns Supplementary files format and content: tables of BZ genes and interspecies comparison of BZ between mouse and zebrafish,adult heart,Unfixed hearts were isolated and tissue was cut out and embedded in tissue freezing medium. Blocks were cryosectioned and individual sections were transferred to Eppendorf tubes on dry ice. For extended experimental procedure see also Junker et al. 2014 Cell.,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,tissue:adult heart|transgenic background:wildtype|section thickness:Took every section|section direction:from injured area into the remote myocardium|treatment:cryoinjury|timepoint:3dpi,GSM7717522,GSM7717522: zebrafish 3dpi heart1; Danio rerio; RNA Seq,GSM7717522 r1,GSM7717522,1,Total RNA was isolated by TRIzol extraction. mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012 see also: Junker et al. 2014 Cell CEL seq protocol Hashimshony et al. Cell Reports 2012; also see: Junker et al. 2014 Cell,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP455780,,,FKHEART1_R1.fastq FKHEART1_R2.fastq,fastq fastq,784558404.0,5209213.0,GSM7717522 r1,0:75.22 1:75.39,A:291009319;C:102718146;G:111606154;T:279144074;N:80711,75,75,,,291009319,102718146,111606154,279144074,80711,SRX21410753,SRS18649235,SRA1751823,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.13119,0.82035,0.12871,0.08445,0.99997,0.82785,0.0,0.56254,76,76,T,B,mate1 technical by mapping diff,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Netherlands,2023-08-18,Adult,Adult,Heart,Cardiovascular System 25239,SRR25723792,SRX21447930,SRS18684553,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 4,GSM7728123,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 4,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728123,GSM7728123: hmga1a mutant plate 4; Danio rerio; RNA Seq,GSM7728123 r1,GSM7728123,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-008_HNWVVBGX9_S7_L001_R2_001.fastq.gz HUB-DB-008_HNWVVBGX9_S7_L001_R1_001.fastq.gz,fastq fastq,529790530.0,6160355.0,GSM7728123 r1,0:26 1:60,A:134254092;C:100685793;G:108463112;T:186279563;N:107970,26,60,,,134254092,100685793,108463112,186279563,107970,SRX21447930,SRS18684553,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11277,0.80912,0.10457,0.43275,0.99131,0.93933,0.67026,0.54274,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25240,SRR25723793,SRX21447930,SRS18684553,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 4,GSM7728123,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 4,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728123,GSM7728123: hmga1a mutant plate 4; Danio rerio; RNA Seq,GSM7728123 r1,GSM7728123,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-008_HNWVVBGX9_S7_L002_R1_001.fastq.gz HUB-DB-008_HNWVVBGX9_S7_L002_R2_001.fastq.gz,fastq fastq,611015122.0,7104827.0,GSM7728123 r2,0:26 1:60,A:152137832;C:116026517;G:127735448;T:214967198;N:148127,26,60,,,152137832,116026517,127735448,214967198,148127,SRX21447930,SRS18684553,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11413,0.81785,0.10565,0.44205,0.99119,0.93419,0.59743,0.53882,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25241,SRR25723794,SRX21447930,SRS18684553,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 4,GSM7728123,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 4,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728123,GSM7728123: hmga1a mutant plate 4; Danio rerio; RNA Seq,GSM7728123 r1,GSM7728123,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-008_HNWVVBGX9_S7_L003_R2_001.fastq.gz HUB-DB-008_HNWVVBGX9_S7_L003_R1_001.fastq.gz,fastq fastq,554981822.0,6453277.0,GSM7728123 r3,0:26 1:60,A:140664360;C:105281601;G:113575873;T:195359632;N:100356,26,60,,,140664360,105281601,113575873,195359632,100356,SRX21447930,SRS18684553,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11352,0.80651,0.10538,0.42971,0.99137,0.93994,0.65893,0.53098,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25242,SRR25723795,SRX21447930,SRS18684553,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 4,GSM7728123,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 4,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728123,GSM7728123: hmga1a mutant plate 4; Danio rerio; RNA Seq,GSM7728123 r1,GSM7728123,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-008_HNWVVBGX9_S7_L004_R2_001.fastq.gz HUB-DB-008_HNWVVBGX9_S7_L004_R1_001.fastq.gz,fastq fastq,459861436.0,5347226.0,GSM7728123 r4,0:26 1:60,A:115949245;C:86913961;G:95720860;T:161187969;N:89401,26,60,,,115949245,86913961,95720860,161187969,89401,SRX21447930,SRS18684553,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11249,0.81109,0.10407,0.43334,0.99135,0.94034,0.64852,0.53864,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25243,SRR25723796,SRX21447929,SRS18684552,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 3,GSM7728122,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728122,GSM7728122: hmga1a mutant plate 3; Danio rerio; RNA Seq,GSM7728122 r1,GSM7728122,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-007_HNWVVBGX9_S6_L001_R1_001.fastq.gz HUB-DB-007_HNWVVBGX9_S6_L001_R2_001.fastq.gz,fastq fastq,584059454.0,6791389.0,GSM7728122 r1,0:26 1:60,A:162038298;C:107975394;G:118251259;T:195669218;N:125285,26,60,,,162038298,107975394,118251259,195669218,125285,SRX21447929,SRS18684552,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.08872,0.68673,0.08328,0.37858,0.99346,0.94945,0.60282,0.42608,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25244,SRR25723797,SRX21447929,SRS18684552,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 3,GSM7728122,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728122,GSM7728122: hmga1a mutant plate 3; Danio rerio; RNA Seq,GSM7728122 r1,GSM7728122,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-007_HNWVVBGX9_S6_L002_R1_001.fastq.gz HUB-DB-007_HNWVVBGX9_S6_L002_R2_001.fastq.gz,fastq fastq,641359448.0,7457668.0,GSM7728122 r2,0:26 1:60,A:174443468;C:118438230;G:132604485;T:215721538;N:151727,26,60,,,174443468,118438230,132604485,215721538,151727,SRX21447929,SRS18684552,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.09277,0.71427,0.08722,0.3942,0.99338,0.94594,0.56903,0.45872,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25245,SRR25723798,SRX21447929,SRS18684552,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 3,GSM7728122,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728122,GSM7728122: hmga1a mutant plate 3; Danio rerio; RNA Seq,GSM7728122 r1,GSM7728122,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-007_HNWVVBGX9_S6_L003_R1_001.fastq.gz HUB-DB-007_HNWVVBGX9_S6_L003_R2_001.fastq.gz,fastq fastq,609857046.0,7091361.0,GSM7728122 r3,0:26 1:60,A:169007241;C:112556048;G:123345017;T:204831635;N:117105,26,60,,,169007241,112556048,123345017,204831635,117105,SRX21447929,SRS18684552,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.09058,0.68251,0.08521,0.37718,0.99332,0.95156,0.59403,0.44605,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25246,SRR25723799,SRX21447929,SRS18684552,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 3,GSM7728122,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728122,GSM7728122: hmga1a mutant plate 3; Danio rerio; RNA Seq,GSM7728122 r1,GSM7728122,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-007_HNWVVBGX9_S6_L004_R1_001.fastq.gz HUB-DB-007_HNWVVBGX9_S6_L004_R2_001.fastq.gz,fastq fastq,514759966.0,5985581.0,GSM7728122 r4,0:26 1:60,A:142060859;C:94579961;G:106100208;T:171916257;N:102681,26,60,,,142060859,94579961,106100208,171916257,102681,SRX21447929,SRS18684552,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.08962,0.7176,0.08411,0.39394,0.99293,0.95189,0.55398,0.46149,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25247,SRR25723800,SRX21447928,SRS18684551,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 3,GSM7728121,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728121,GSM7728121: wildtype plate 3; Danio rerio; RNA Seq,GSM7728121 r1,GSM7728121,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-006_HNWVVBGX9_S5_L001_R1_001.fastq.gz HUB-DB-006_HNWVVBGX9_S5_L001_R2_001.fastq.gz,fastq fastq,929999528.0,10813948.0,GSM7728121 r1,0:26 1:60,A:240334465;C:174255952;G:184260587;T:330943403;N:205121,26,60,,,240334465,174255952,184260587,330943403,205121,SRX21447928,SRS18684551,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11113,0.87407,0.10304,0.43976,0.992,0.93034,0.63031,0.44624,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25248,SRR25723801,SRX21447928,SRS18684551,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 3,GSM7728121,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728121,GSM7728121: wildtype plate 3; Danio rerio; RNA Seq,GSM7728121 r1,GSM7728121,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-006_HNWVVBGX9_S5_L002_R1_001.fastq.gz HUB-DB-006_HNWVVBGX9_S5_L002_R2_001.fastq.gz,fastq fastq,987824896.0,11486336.0,GSM7728121 r2,0:26 1:60,A:251121714;C:184449811;G:201317783;T:350692225;N:243363,26,60,,,251121714,184449811,201317783,350692225,243363,SRX21447928,SRS18684551,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11267,0.87874,0.10429,0.4457,0.99184,0.92498,0.56612,0.45489,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25249,SRR25723802,SRX21447928,SRS18684551,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 3,GSM7728121,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728121,GSM7728121: wildtype plate 3; Danio rerio; RNA Seq,GSM7728121 r1,GSM7728121,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-006_HNWVVBGX9_S5_L003_R1_001.fastq.gz HUB-DB-006_HNWVVBGX9_S5_L003_R2_001.fastq.gz,fastq fastq,959933032.0,11162012.0,GSM7728121 r3,0:26 1:60,A:247983556;C:179564753;G:190342582;T:341858456;N:183685,26,60,,,247983556,179564753,190342582,341858456,183685,SRX21447928,SRS18684551,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.1118,0.87156,0.10348,0.43983,0.99149,0.93034,0.65287,0.44548,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25250,SRR25723803,SRX21447928,SRS18684551,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 3,GSM7728121,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 3,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728121,GSM7728121: wildtype plate 3; Danio rerio; RNA Seq,GSM7728121 r1,GSM7728121,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-006_HNWVVBGX9_S5_L004_R1_001.fastq.gz HUB-DB-006_HNWVVBGX9_S5_L004_R2_001.fastq.gz,fastq fastq,798370766.0,9283381.0,GSM7728121 r4,0:26 1:60,A:205538880;C:148510556;G:161196381;T:282964175;N:160774,26,60,,,205538880,148510556,161196381,282964175,160774,SRX21447928,SRS18684551,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11018,0.87017,0.10214,0.43625,0.99226,0.93332,0.56058,0.44211,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25251,SRR25723804,SRX21447927,SRS18684550,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 2,GSM7728120,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728120,GSM7728120: wildtype plate 2; Danio rerio; RNA Seq,GSM7728120 r1,GSM7728120,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-005_HNWVVBGX9_S4_L001_R1_001.fastq.gz HUB-DB-005_HNWVVBGX9_S4_L001_R2_001.fastq.gz,fastq fastq,806178534.0,9374169.0,GSM7728120 r1,0:26 1:60,A:213590554;C:148614157;G:163804315;T:279996916;N:172592,26,60,,,213590554,148614157,163804315,279996916,172592,SRX21447927,SRS18684550,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10778,0.79219,0.10092,0.39443,0.99255,0.93712,0.64412,0.39504,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25252,SRR25723805,SRX21447927,SRS18684550,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 2,GSM7728120,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728120,GSM7728120: wildtype plate 2; Danio rerio; RNA Seq,GSM7728120 r1,GSM7728120,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-005_HNWVVBGX9_S4_L002_R1_001.fastq.gz HUB-DB-005_HNWVVBGX9_S4_L002_R2_001.fastq.gz,fastq fastq,874594286.0,10169701.0,GSM7728120 r2,0:26 1:60,A:227213153;C:160851586;G:182364146;T:303951184;N:214217,26,60,,,227213153,160851586,182364146,303951184,214217,SRX21447927,SRS18684550,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11064,0.80317,0.1038,0.40093,0.99249,0.9332,0.652,0.40914,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25253,SRR25723806,SRX21447927,SRS18684550,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 2,GSM7728120,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728120,GSM7728120: wildtype plate 2; Danio rerio; RNA Seq,GSM7728120 r1,GSM7728120,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-005_HNWVVBGX9_S4_L003_R1_001.fastq.gz HUB-DB-005_HNWVVBGX9_S4_L003_R2_001.fastq.gz,fastq fastq,831835860.0,9672510.0,GSM7728120 r3,0:26 1:60,A:220300204;C:153061412;G:168938657;T:289375449;N:160138,26,60,,,220300204,153061412,168938657,289375449,160138,SRX21447927,SRS18684550,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10987,0.78665,0.10348,0.39264,0.99316,0.93661,0.66753,0.37676,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25254,SRR25723807,SRX21447927,SRS18684550,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 2,GSM7728120,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728120,GSM7728120: wildtype plate 2; Danio rerio; RNA Seq,GSM7728120 r1,GSM7728120,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-005_HNWVVBGX9_S4_L004_R2_001.fastq.gz HUB-DB-005_HNWVVBGX9_S4_L004_R1_001.fastq.gz,fastq fastq,692573136.0,8053176.0,GSM7728120 r4,0:26 1:60,A:182418113;C:126757471;G:143869511;T:239393143;N:134898,26,60,,,182418113,126757471,143869511,239393143,134898,SRX21447927,SRS18684550,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10825,0.81365,0.10178,0.40513,0.99308,0.94032,0.67299,0.41125,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25255,SRR25723808,SRX21447926,SRS18684549,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 2,GSM7728119,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728119,GSM7728119: hmga1a mutant plate 2; Danio rerio; RNA Seq,GSM7728119 r1,GSM7728119,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-004_AHVKTGBGX7_S7_L001_R1_001.fastq.gz HUB-DB-004_AHVKTGBGX7_S7_L001_R2_001.fastq.gz,fastq fastq,364147736.0,4234276.0,GSM7728119 r1,0:26 1:60,A:94538754;C:69024492;G:68361658;T:132108181;N:114651,26,60,,,94538754,69024492,68361658,132108181,114651,SRX21447926,SRS18684549,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11558,0.78278,0.10659,0.24226,0.9893,0.92719,0.58692,0.48105,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25256,SRR25723809,SRX21447926,SRS18684549,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 2,GSM7728119,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728119,GSM7728119: hmga1a mutant plate 2; Danio rerio; RNA Seq,GSM7728119 r1,GSM7728119,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-004_AHVKTGBGX7_S7_L002_R1_001.fastq.gz HUB-DB-004_AHVKTGBGX7_S7_L002_R2_001.fastq.gz,fastq fastq,377653262.0,4391317.0,GSM7728119 r2,0:26 1:60,A:99999342;C:71104204;G:70005970;T:136434888;N:108858,26,60,,,99999342,71104204,70005970,136434888,108858,SRX21447926,SRS18684549,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11405,0.77996,0.10525,0.24008,0.98963,0.93616,0.60855,0.44129,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25257,SRR25723810,SRX21447926,SRS18684549,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 2,GSM7728119,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728119,GSM7728119: hmga1a mutant plate 2; Danio rerio; RNA Seq,GSM7728119 r1,GSM7728119,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-004_AHVKTGBGX7_S7_L003_R1_001.fastq.gz HUB-DB-004_AHVKTGBGX7_S7_L003_R2_001.fastq.gz,fastq fastq,306947588.0,3569158.0,GSM7728119 r3,0:26 1:60,A:80261116;C:58220837;G:57196503;T:111195958;N:73174,26,60,,,80261116,58220837,57196503,111195958,73174,SRX21447926,SRS18684549,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11141,0.76713,0.10254,0.23772,0.98938,0.93314,0.62261,0.47885,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25258,SRR25723811,SRX21447926,SRS18684549,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 2,GSM7728119,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 2,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728119,GSM7728119: hmga1a mutant plate 2; Danio rerio; RNA Seq,GSM7728119 r1,GSM7728119,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-004_AHVKTGBGX7_S7_L004_R1_001.fastq.gz HUB-DB-004_AHVKTGBGX7_S7_L004_R2_001.fastq.gz,fastq fastq,389472414.0,4528749.0,GSM7728119 r4,0:26 1:60,A:102486709;C:73105388;G:73019179;T:140769931;N:91207,26,60,,,102486709,73105388,73019179,140769931,91207,SRX21447926,SRS18684549,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11502,0.78307,0.10673,0.24219,0.98967,0.93308,0.64527,0.43659,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25259,SRR25723812,SRX21447925,SRS18684547,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 1,GSM7728118,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728118,GSM7728118: hmga1a mutant plate 1; Danio rerio; RNA Seq,GSM7728118 r1,GSM7728118,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-003_AHVKTGBGX7_S6_L001_R1_001.fastq.gz HUB-DB-003_AHVKTGBGX7_S6_L001_R2_001.fastq.gz,fastq fastq,381365624.0,4434484.0,GSM7728118 r1,0:26 1:60,A:102130758;C:71032768;G:73641442;T:134437630;N:123026,26,60,,,102130758,71032768,73641442,134437630,123026,SRX21447925,SRS18684547,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10052,0.7243,0.09214,0.25633,0.9892,0.92985,0.52345,0.5112,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25260,SRR25723813,SRX21447925,SRS18684547,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 1,GSM7728118,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728118,GSM7728118: hmga1a mutant plate 1; Danio rerio; RNA Seq,GSM7728118 r1,GSM7728118,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-003_AHVKTGBGX7_S6_L002_R1_001.fastq.gz HUB-DB-003_AHVKTGBGX7_S6_L002_R2_001.fastq.gz,fastq fastq,400525220.0,4657270.0,GSM7728118 r2,0:26 1:60,A:109307778;C:73934198;G:76916762;T:140249156;N:117326,26,60,,,109307778,73934198,76916762,140249156,117326,SRX21447925,SRS18684547,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.09923,0.71601,0.09078,0.25139,0.98948,0.93914,0.52314,0.5078,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25261,SRR25723814,SRX21447925,SRS18684547,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 1,GSM7728118,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728118,GSM7728118: hmga1a mutant plate 1; Danio rerio; RNA Seq,GSM7728118 r1,GSM7728118,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-003_AHVKTGBGX7_S6_L003_R1_001.fastq.gz HUB-DB-003_AHVKTGBGX7_S6_L003_R2_001.fastq.gz,fastq fastq,341364014.0,3969349.0,GSM7728118 r3,0:26 1:60,A:91716565;C:63676514;G:65563102;T:120323617;N:84216,26,60,,,91716565,63676514,65563102,120323617,84216,SRX21447925,SRS18684547,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.09826,0.7079,0.09008,0.2497,0.98918,0.93809,0.52649,0.49781,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25262,SRR25723815,SRX21447925,SRS18684547,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,hmga1a mutant plate 1,GSM7728118,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,hmga1a mutant plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:hmga1a mutant|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728118,GSM7728118: hmga1a mutant plate 1; Danio rerio; RNA Seq,GSM7728118 r1,GSM7728118,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-003_AHVKTGBGX7_S6_L004_R1_001.fastq.gz HUB-DB-003_AHVKTGBGX7_S6_L004_R2_001.fastq.gz,fastq fastq,414000732.0,4813962.0,GSM7728118 r4,0:26 1:60,A:112224009;C:76213738;G:80259829;T:145201785;N:101371,26,60,,,112224009,76213738,80259829,145201785,101371,SRX21447925,SRS18684547,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10103,0.71981,0.09294,0.25413,0.98902,0.93669,0.52994,0.51172,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25263,SRR25723816,SRX21447924,SRS18684548,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 1,GSM7728117,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728117,GSM7728117: wildtype plate 1; Danio rerio; RNA Seq,GSM7728117 r1,GSM7728117,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-001_AHVKTGBGX7_S5_L001_R1_001.fastq.gz HUB-DB-001_AHVKTGBGX7_S5_L001_R2_001.fastq.gz,fastq fastq,471703034.0,5484919.0,GSM7728117 r1,0:26 1:60,A:125045012;C:89627491;G:85413855;T:171466861;N:149815,26,60,,,125045012,89627491,85413855,171466861,149815,SRX21447924,SRS18684548,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10906,0.68002,0.10212,0.30506,0.99101,0.94219,0.63738,0.45862,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25264,SRR25723817,SRX21447924,SRS18684548,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 1,GSM7728117,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728117,GSM7728117: wildtype plate 1; Danio rerio; RNA Seq,GSM7728117 r1,GSM7728117,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-001_AHVKTGBGX7_S5_L002_R1_001.fastq.gz HUB-DB-001_AHVKTGBGX7_S5_L002_R2_001.fastq.gz,fastq fastq,489372938.0,5690383.0,GSM7728117 r2,0:26 1:60,A:131516821;C:92202683;G:88685608;T:176824985;N:142841,26,60,,,131516821,92202683,88685608,176824985,142841,SRX21447924,SRS18684548,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.11042,0.68086,0.10299,0.30411,0.99093,0.94856,0.60224,0.46578,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25265,SRR25723818,SRX21447924,SRS18684548,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 1,GSM7728117,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728117,GSM7728117: wildtype plate 1; Danio rerio; RNA Seq,GSM7728117 r1,GSM7728117,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-001_AHVKTGBGX7_S5_L003_R1_001.fastq.gz HUB-DB-001_AHVKTGBGX7_S5_L003_R2_001.fastq.gz,fastq fastq,407735288.0,4741108.0,GSM7728117 r3,0:26 1:60,A:108841476;C:77418215;G:73171345;T:148203998;N:100254,26,60,,,108841476,77418215,73171345,148203998,100254,SRX21447924,SRS18684548,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10882,0.67068,0.10177,0.29557,0.99151,0.94686,0.65762,0.42658,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 25266,SRR25723819,SRX21447924,SRS18684548,SRP456323,PRJNA1008049,Interspecies comparison reveals Hmga1 as driver of cardiac regeneration [scRNA seq],GSE241390,Transcriptome Analysis,The prospect of repairing the heart post a myocardial infarction by promoting cardiomyocyte proliferation has gained momentum from studies showing the heart's regenerative ability in fish amphibians and neonatal mammals. Despite evidence of varying cardiomyocyte proliferation rates among species the molecular mechanisms driving cardiomyocyte cell cycle re entry remain insufficiently understood. In this study we employed spatial transcriptomics and identified high mobility group AT hook 1a Hmga1a as being upregulated in cardiomyocytes of the injury border zone in zebrafish but not in mice. Through knock out and cardiomyocyte specific overexpression of hmga1a we found that Hmga1a was required for zebrafish heart regeneration and sufficient to drive cardiomyocyte proliferation. In addition a single injection of Hmga1 virus in injured mouse hearts resulted in increased border zone cardiomyocyte proliferation and improved heart function. Mechanistically Hmga1 expression reduced repressive H3K27me3 histone modifications from developmentally regulated genes and induced a border zone like transcriptional program in adult cardiomyocytes. Our study demonstrates the value of interspecies comparisons by identifying Hmga1 as a critical driver of heart regeneration and highlights Hmga1 as a promising therapeutic candidate to improve cardiac xxx post injury. Overall design: TOMOseq was performed on mouse and zebrafish hearts at 3 7 and 14 xxx post injury for the interspecies comparison. single cell RNA sequencing was performed on wildtype versus hmga1a mutant zebrafish cardiomyocytes 7 days post cryoinjury to assess differences during heart regeneration. Bulk RNA sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect in an uninjured heart. bulk sortChIC sequencing was performed on control versus Hmga1a overexpression cardiomyocytes to assess the effect of Hmga1a on the chromatin.,parent bioproject:PRJNA1006653,pubmed:39747457,,wildtype plate 1,GSM7728117,,source name:adult heart|tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury|geo loc name:missing|collection date:missing,wildtype plate 1,Based on the distribution of the log10 total reads plotted against the frequency we introduced a cutoff at minimally 600 reads per cell to be included for further analysis which left us with a total of 1310 cells 653 wildtype and 657 hmga1a / cells. Next single cell data was analyzed using Seurat 81. Using the following parameters: Variable features = all genes dimensions = 8 resolution = 0.7. This resulted in the identification of 6 clusters which were plotted in a two dimensional UMAP. Monocle2 was used to identify genes that were differentially expressed over pseudo time and organized in a self organizing map with of 8 modules. Pseudo temporal values assigned to single cells were integrated in our Seurat object and the pseudo temporal ordering was plotted on the UMAP. DAVID was used to perform GO analysis for the 8 modules. Assembly: danrer9 Supplementary files format and content: excel file containing pseudotime modules and their genes and GO terms per module Supplementary files format and content: count tables transcriptcounts,adult heart,cryoinjury was performed 7 days before hearts were extracted,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,tissue:adult heart|genotype:nppa:mCitrine|genotype:wildtype|cell type:border z1 cardiomyocytes|treatment:7 days post cryoinjury,GSM7728117,GSM7728117: wildtype plate 1; Danio rerio; RNA Seq,GSM7728117 r1,GSM7728117,1,Unfixed ventricles were dissociated by Collagenase and TrypLE Express treatment to prepare single cell solutions. Cells were subsequently FACS sorted based on nppa:mCitrine expression in 384 well plates each well containing 1 cell containing Vapor Lock oil with CEL Seq2 barcoded primers spike in mRNA and dNTPs Single Cell Discoveries. Plates were spun down and frozen at 80oC until ready for library prep. Single cell sequencing libraries were prepared by Single Cell Discoveries Utrecht The Netherlands using the SORT seq protocol . The CEL Seq2 protocol was used for library prep Hashimshony et al. 2016. Illumina sequencing libraries were prepared with the TruSeq small RNA primers Illumina and paired end sequenced at 75 bp read length on the Illumina NextSeq platform. In total seven 384 well plates were sequenced containing one cell per well of which three were obtained from wildtype cells and four from hmga1a mutant cells.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP456323,,,HUB-DB-001_AHVKTGBGX7_S5_L004_R1_001.fastq.gz HUB-DB-001_AHVKTGBGX7_S5_L004_R2_001.fastq.gz,fastq fastq,505539304.0,5878364.0,GSM7728117 r4,0:26 1:60,A:135189083;C:94908231;G:92431583;T:182885125;N:125282,26,60,,,135189083,94908231,92431583,182885125,125282,SRX21447924,SRS18684548,SRA1698978,"Jeroen Bakkers, Hubrecht Institute","Jeroen Bakkers, Hubrecht Institute",2,0.10989,0.68603,0.10286,0.30542,0.99107,0.94777,0.65338,0.42121,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,Netherlands,2023-08-22,Adult,Adult,Heart,Cardiovascular System 30508,SRR27764648,SRX23429609,SRS20284060,SRP486518,PRJNA1070582,Gene expression profile of neighboring cells of cells with unfit Wnt morphogen gradient during cell competition,GSE254439,Transcriptome Analysis,Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength dependent manner to pattern developing tissues. However developing tissues also undergo dynamic morphogenesis which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradient. Here we show that a cell competition related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß catenin signalling gradient which acts as a morphogen to establish embryonic anterior posterior patterning revealed that unfit cells with abnormal Wnt/ß catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß catenin gradient formation and consequent anterior posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues this system may support tissue robustness and disease prevention. Overall design: ß catCA constitutive active form of ß catenin expressing GFP+ cells or ßcatCA unexpressing GFP cells from ß catCA mosaically introduced zebrafish early embryos were sorted by FACS cell sorter. Total RNAs were extracted and analyzed by RNA seq.,,pubmed:39546611,,Mosaic GFP control GFP min lot2,GSM8042248,,source name:early embryonic cell|strain:AB|tissue:early embryonic cell|developmental stage:9 hpf loc name:missing|collection date:missing,Mosaic GFP control GFP min lot2,Sequenced reads were mapped to the GRCz10 reference genome using Bowtie2 ver. 2.3.1. UMI counts were extracted per HTSeq ver. 0.6.1 from the CEL Seq2 pipeline. Assembly: GRCz10 Supplementary files format and content: Tab delimited text file for expression data normalized by regularized logarithm.,early embryonic cell,,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,strain:AB|tissue:early embryonic cell|developmental stage:9 hpf,GSM8042248,GSM8042248: Mosaic GFP control GFP min lot2; Danio rerio; RNA Seq,GSM8042248 r1,GSM8042248,1,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 1500,,SRP486518,,,Mosaic_GFP_control_GFP_min_lot2.fastq.gz,fastq,7474464.0,207624.0,GSM8042248 r1,0:36,A:1796571;C:1274347;G:1548876;T:2854637;N:33,36,,,,1796571,1274347,1548876,2854637,33,SRX23429609,SRS20284060,SRA1793856,"Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University","Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University",1,0.82922,,0.20207,,0.86143,,0.54661,,36,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Japan,2024-01-29,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 30509,SRR27764649,SRX23429608,SRS20284059,SRP486518,PRJNA1070582,Gene expression profile of neighboring cells of cells with unfit Wnt morphogen gradient during cell competition,GSE254439,Transcriptome Analysis,Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength dependent manner to pattern developing tissues. However developing tissues also undergo dynamic morphogenesis which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradient. Here we show that a cell competition related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß catenin signalling gradient which acts as a morphogen to establish embryonic anterior posterior patterning revealed that unfit cells with abnormal Wnt/ß catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß catenin gradient formation and consequent anterior posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues this system may support tissue robustness and disease prevention. Overall design: ß catCA constitutive active form of ß catenin expressing GFP+ cells or ßcatCA unexpressing GFP cells from ß catCA mosaically introduced zebrafish early embryos were sorted by FACS cell sorter. Total RNAs were extracted and analyzed by RNA seq.,,pubmed:39546611,,Mosaic GFP control GFP min lot1,GSM8042247,,source name:early embryonic cell|strain:AB|tissue:early embryonic cell|developmental stage:9 hpf loc name:missing|collection date:missing,Mosaic GFP control GFP min lot1,Sequenced reads were mapped to the GRCz10 reference genome using Bowtie2 ver. 2.3.1. UMI counts were extracted per HTSeq ver. 0.6.1 from the CEL Seq2 pipeline. Assembly: GRCz10 Supplementary files format and content: Tab delimited text file for expression data normalized by regularized logarithm.,early embryonic cell,,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,strain:AB|tissue:early embryonic cell|developmental stage:9 hpf,GSM8042247,GSM8042247: Mosaic GFP control GFP min lot1; Danio rerio; RNA Seq,GSM8042247 r1,GSM8042247,1,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 1500,,SRP486518,,,Mosaic_GFP_control_GFP_min_lot1.fastq.gz,fastq,8256240.0,229340.0,GSM8042247 r1,0:36,A:1947003;C:1345938;G:1692065;T:3271133;N:101,36,,,,1947003,1345938,1692065,3271133,101,SRX23429608,SRS20284059,SRA1793856,"Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University","Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University",1,0.83305,,0.17754,,0.8619,,0.61474,,36,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Japan,2024-01-29,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 30510,SRR27764650,SRX23429607,SRS20284058,SRP486518,PRJNA1070582,Gene expression profile of neighboring cells of cells with unfit Wnt morphogen gradient during cell competition,GSE254439,Transcriptome Analysis,Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength dependent manner to pattern developing tissues. However developing tissues also undergo dynamic morphogenesis which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradient. Here we show that a cell competition related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß catenin signalling gradient which acts as a morphogen to establish embryonic anterior posterior patterning revealed that unfit cells with abnormal Wnt/ß catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß catenin gradient formation and consequent anterior posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues this system may support tissue robustness and disease prevention. Overall design: ß catCA constitutive active form of ß catenin expressing GFP+ cells or ßcatCA unexpressing GFP cells from ß catCA mosaically introduced zebrafish early embryos were sorted by FACS cell sorter. Total RNAs were extracted and analyzed by RNA seq.,,pubmed:39546611,,Mosaic beta catCA GFP min lot2,GSM8042246,,source name:early embryonic cell|strain:AB|tissue:early embryonic cell|developmental stage:9 hpf loc name:missing|collection date:missing,Mosaic beta catCA GFP min lot2,Sequenced reads were mapped to the GRCz10 reference genome using Bowtie2 ver. 2.3.1. UMI counts were extracted per HTSeq ver. 0.6.1 from the CEL Seq2 pipeline. Assembly: GRCz10 Supplementary files format and content: Tab delimited text file for expression data normalized by regularized logarithm.,early embryonic cell,,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,strain:AB|tissue:early embryonic cell|developmental stage:9 hpf,GSM8042246,GSM8042246: Mosaic beta catCA GFP min lot2; Danio rerio; RNA Seq,GSM8042246 r1,GSM8042246,1,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 1500,,SRP486518,,,Mosaic_beta-catCA_GFP_min_lot2.fastq.gz,fastq,856836.0,23801.0,GSM8042246 r1,0:36,A:217589;C:147419;G:175612;T:316201;N:15,36,,,,217589,147419,175612,316201,15,SRX23429607,SRS20284058,SRA1793856,"Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University","Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University",1,0.82481,,0.28434,,0.95371,,0.61551,,36,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Japan,2024-01-29,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 30511,SRR27764651,SRX23429606,SRS20284057,SRP486518,PRJNA1070582,Gene expression profile of neighboring cells of cells with unfit Wnt morphogen gradient during cell competition,GSE254439,Transcriptome Analysis,Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength dependent manner to pattern developing tissues. However developing tissues also undergo dynamic morphogenesis which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradient. Here we show that a cell competition related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß catenin signalling gradient which acts as a morphogen to establish embryonic anterior posterior patterning revealed that unfit cells with abnormal Wnt/ß catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß catenin gradient formation and consequent anterior posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues this system may support tissue robustness and disease prevention. Overall design: ß catCA constitutive active form of ß catenin expressing GFP+ cells or ßcatCA unexpressing GFP cells from ß catCA mosaically introduced zebrafish early embryos were sorted by FACS cell sorter. Total RNAs were extracted and analyzed by RNA seq.,,pubmed:39546611,,Mosaic beta catCA GFP min lot1,GSM8042245,,source name:early embryonic cell|strain:AB|tissue:early embryonic cell|developmental stage:9 hpf loc name:missing|collection date:missing,Mosaic beta catCA GFP min lot1,Sequenced reads were mapped to the GRCz10 reference genome using Bowtie2 ver. 2.3.1. UMI counts were extracted per HTSeq ver. 0.6.1 from the CEL Seq2 pipeline. Assembly: GRCz10 Supplementary files format and content: Tab delimited text file for expression data normalized by regularized logarithm.,early embryonic cell,,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,strain:AB|tissue:early embryonic cell|developmental stage:9 hpf,GSM8042245,GSM8042245: Mosaic beta catCA GFP min lot1; Danio rerio; RNA Seq,GSM8042245 r1,GSM8042245,1,Cells were dissolved in TRIzol reagent Invitrogen and extracted. Samples were prepared according to the CEL Seq2 protocol described in Hashimshony et al. Geneome Biology 2016. Libraries preparation was performed according to the published CEL Seq2 protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 1500,,SRP486518,,,Mosaic_beta-catCA_GFP_min_lot1.fastq.gz,fastq,7181388.0,199483.0,GSM8042245 r1,0:36,A:1744919;C:1207643;G:1456829;T:2771919;N:78,36,,,,1744919,1207643,1456829,2771919,78,SRX23429606,SRS20284057,SRA1793856,"Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University","Department of Homeostatic Regulation, Research Institute for Microbial Diseases, Osaka University",1,0.83629,,0.16823,,0.84774,,0.64548,,36,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Japan,2024-01-29,Gastrula,Embryo,Embryo Imprecise,All anatomical structures 33075,SRR29654109,SRX25158199,SRS21848821,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,GSM8369980,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369980,GSM8369980: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369980 r1,GSM8369980,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_AMD_-016_cbc.fastq.gz,fastq,347162880.0,5786048.0,GSM8369980 r1,0:60,A:122562825;C:62219285;G:75539895;T:86775172;N:65703,60,,,,122562825,62219285,75539895,86775172,65703,SRX25158199,SRS21848821,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33076,SRR29654110,SRX25158199,SRS21848821,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,GSM8369980,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369980,GSM8369980: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369980 r1,GSM8369980,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_AMD_-016_cbc.fastq.gz,fastq,340954260.0,5682571.0,GSM8369980 r2,0:60,A:120149623;C:60854408;G:75192893;T:84703966;N:53370,60,,,,120149623,60854408,75192893,84703966,53370,SRX25158199,SRS21848821,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33077,SRR29654111,SRX25158199,SRS21848821,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,GSM8369980,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369980,GSM8369980: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369980 r1,GSM8369980,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_AMD_-016_cbc.fastq.gz,fastq,351749160.0,5862486.0,GSM8369980 r3,0:60,A:124352905;C:63078724;G:76291828;T:87988386;N:37317,60,,,,124352905,63078724,76291828,87988386,37317,SRX25158199,SRS21848821,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33078,SRR29654112,SRX25158199,SRS21848821,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,GSM8369980,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369980,GSM8369980: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369980 r1,GSM8369980,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_AMD_-016_cbc.fastq.gz,fastq,345225720.0,5753762.0,GSM8369980 r4,0:60,A:121803902;C:61651407;G:75900430;T:85843789;N:26192,60,,,,121803902,61651407,75900430,85843789,26192,SRX25158199,SRS21848821,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33079,SRR29654113,SRX25158198,SRS21848820,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,GSM8369979,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369979,GSM8369979: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369979 r1,GSM8369979,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_AMD_-015_cbc.fastq.gz,fastq,159497640.0,2658294.0,GSM8369979 r1,0:60,A:55569742;C:28678683;G:33264744;T:41953354;N:31117,60,,,,55569742,28678683,33264744,41953354,31117,SRX25158198,SRS21848820,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33080,SRR29654114,SRX25158198,SRS21848820,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,GSM8369979,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369979,GSM8369979: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369979 r1,GSM8369979,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_AMD_-015_cbc.fastq.gz,fastq,156549240.0,2609154.0,GSM8369979 r2,0:60,A:54428858;C:28027289;G:33149650;T:40918247;N:25196,60,,,,54428858,28027289,33149650,40918247,25196,SRX25158198,SRS21848820,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33081,SRR29654115,SRX25158198,SRS21848820,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,GSM8369979,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369979,GSM8369979: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369979 r1,GSM8369979,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_AMD_-015_cbc.fastq.gz,fastq,161735280.0,2695588.0,GSM8369979 r3,0:60,A:56425187;C:29084972;G:33637112;T:42570603;N:17406,60,,,,56425187,29084972,33637112,42570603,17406,SRX25158198,SRS21848820,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33082,SRR29654116,SRX25158198,SRS21848820,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,GSM8369979,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369979,GSM8369979: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369979 r1,GSM8369979,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_AMD_-015_cbc.fastq.gz,fastq,158621520.0,2643692.0,GSM8369979 r4,0:60,A:55213721;C:28430013;G:33462307;T:41503213;N:12266,60,,,,55213721,28430013,33462307,41503213,12266,SRX25158198,SRS21848820,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33083,SRR29654117,SRX25158197,SRS21848819,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,GSM8369978,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369978,GSM8369978: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369978 r1,GSM8369978,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_AMD_-011_cbc.fastq.gz,fastq,44363040.0,739384.0,GSM8369978 r1,0:60,A:17756825;C:7540147;G:9311367;T:9746444;N:8257,60,,,,17756825,7540147,9311367,9746444,8257,SRX25158197,SRS21848819,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33084,SRR29654118,SRX25158197,SRS21848819,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,GSM8369978,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369978,GSM8369978: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369978 r1,GSM8369978,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_AMD_-011_cbc.fastq.gz,fastq,43450800.0,724180.0,GSM8369978 r2,0:60,A:17376442;C:7355139;G:9244019;T:9468455;N:6745,60,,,,17376442,7355139,9244019,9468455,6745,SRX25158197,SRS21848819,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33085,SRR29654119,SRX25158197,SRS21848819,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,GSM8369978,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369978,GSM8369978: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369978 r1,GSM8369978,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_AMD_-011_cbc.fastq.gz,fastq,44795880.0,746598.0,GSM8369978 r3,0:60,A:17988707;C:7600964;G:9362568;T:9839285;N:4356,60,,,,17988707,7600964,9362568,9839285,4356,SRX25158197,SRS21848819,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33086,SRR29654120,SRX25158197,SRS21848819,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,GSM8369978,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury AMD3100,GSM8369978,GSM8369978: Neutrophils mpx+ 8 xxx post injury AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369978 r1,GSM8369978,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_AMD_-011_cbc.fastq.gz,fastq,44060940.0,734349.0,GSM8369978 r4,0:60,A:17608648;C:7492643;G:9327676;T:9628642;N:3331,60,,,,17608648,7492643,9327676,9628642,3331,SRX25158197,SRS21848819,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33087,SRR29654121,SRX25158196,SRS21848818,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 3,GSM8369977,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369977,GSM8369977: Neutrophils mpx+ uninjured AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369977 r1,GSM8369977,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_AMD_-014_cbc.fastq.gz,fastq,115048140.0,1917469.0,GSM8369977 r1,0:60,A:41373215;C:21759509;G:21832537;T:30061744;N:21135,60,,,,41373215,21759509,21832537,30061744,21135,SRX25158196,SRS21848818,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33088,SRR29654122,SRX25158196,SRS21848818,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 3,GSM8369977,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369977,GSM8369977: Neutrophils mpx+ uninjured AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369977 r1,GSM8369977,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_AMD_-014_cbc.fastq.gz,fastq,112845060.0,1880751.0,GSM8369977 r2,0:60,A:40482360;C:21287713;G:21750928;T:29306465;N:17594,60,,,,40482360,21287713,21750928,29306465,17594,SRX25158196,SRS21848818,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33089,SRR29654123,SRX25158196,SRS21848818,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 3,GSM8369977,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369977,GSM8369977: Neutrophils mpx+ uninjured AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369977 r1,GSM8369977,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_AMD_-014_cbc.fastq.gz,fastq,116696580.0,1944943.0,GSM8369977 r3,0:60,A:42022776;C:22106040;G:22075778;T:30479513;N:12473,60,,,,42022776,22106040,22075778,30479513,12473,SRX25158196,SRS21848818,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33090,SRR29654124,SRX25158196,SRS21848818,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 3,GSM8369977,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369977,GSM8369977: Neutrophils mpx+ uninjured AMD3100 biol rep 3; Danio rerio; RNA Seq,GSM8369977 r1,GSM8369977,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_AMD_-014_cbc.fastq.gz,fastq,114334980.0,1905583.0,GSM8369977 r4,0:60,A:41132105;C:21578779;G:21934073;T:29681512;N:8511,60,,,,41132105,21578779,21934073,29681512,8511,SRX25158196,SRS21848818,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33091,SRR29654125,SRX25158195,SRS21848817,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 2,GSM8369976,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369976,GSM8369976: Neutrophils mpx+ uninjured AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369976 r1,GSM8369976,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_AMD_-013_cbc.fastq.gz,fastq,354085380.0,5901423.0,GSM8369976 r1,0:60,A:124800871;C:65627966;G:72434440;T:91152029;N:70074,60,,,,124800871,65627966,72434440,91152029,70074,SRX25158195,SRS21848817,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33092,SRR29654126,SRX25158195,SRS21848817,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 2,GSM8369976,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369976,GSM8369976: Neutrophils mpx+ uninjured AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369976 r1,GSM8369976,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_AMD_-013_cbc.fastq.gz,fastq,347250360.0,5787506.0,GSM8369976 r2,0:60,A:122147080;C:64042522;G:72135325;T:88869927;N:55506,60,,,,122147080,64042522,72135325,88869927,55506,SRX25158195,SRS21848817,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33093,SRR29654127,SRX25158195,SRS21848817,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 2,GSM8369976,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369976,GSM8369976: Neutrophils mpx+ uninjured AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369976 r1,GSM8369976,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_AMD_-013_cbc.fastq.gz,fastq,358862580.0,5981043.0,GSM8369976 r3,0:60,A:126636264;C:66527902;G:73214426;T:92445348;N:38640,60,,,,126636264,66527902,73214426,92445348,38640,SRX25158195,SRS21848817,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33094,SRR29654128,SRX25158195,SRS21848817,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 2,GSM8369976,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369976,GSM8369976: Neutrophils mpx+ uninjured AMD3100 biol rep 2; Danio rerio; RNA Seq,GSM8369976 r1,GSM8369976,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_AMD_-013_cbc.fastq.gz,fastq,351843120.0,5864052.0,GSM8369976 r4,0:60,A:123921807;C:64956560;G:72828989;T:90107944;N:27820,60,,,,123921807,64956560,72828989,90107944,27820,SRX25158195,SRS21848817,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33095,SRR29654129,SRX25158194,SRS21848816,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 1,GSM8369975,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369975,GSM8369975: Neutrophils mpx+ uninjured AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369975 r1,GSM8369975,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_AMD_-010_cbc.fastq.gz,fastq,100732380.0,1678873.0,GSM8369975 r1,0:60,A:37215898;C:18610797;G:18111317;T:26774380;N:19988,60,,,,37215898,18610797,18111317,26774380,19988,SRX25158194,SRS21848816,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33096,SRR29654130,SRX25158194,SRS21848816,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 1,GSM8369975,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369975,GSM8369975: Neutrophils mpx+ uninjured AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369975 r1,GSM8369975,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_AMD_-010_cbc.fastq.gz,fastq,98830860.0,1647181.0,GSM8369975 r2,0:60,A:36388012;C:18195984;G:18122717;T:26108692;N:15455,60,,,,36388012,18195984,18122717,26108692,15455,SRX25158194,SRS21848816,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33097,SRR29654131,SRX25158194,SRS21848816,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 1,GSM8369975,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369975,GSM8369975: Neutrophils mpx+ uninjured AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369975 r1,GSM8369975,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_AMD_-010_cbc.fastq.gz,fastq,102277560.0,1704626.0,GSM8369975 r3,0:60,A:37811843;C:18928051;G:18347174;T:27179477;N:11015,60,,,,37811843,18928051,18347174,27179477,11015,SRX25158194,SRS21848816,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33098,SRR29654132,SRX25158194,SRS21848816,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured AMD3100 biol rep 1,GSM8369975,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured AMD3100 biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured AMD3100,GSM8369975,GSM8369975: Neutrophils mpx+ uninjured AMD3100 biol rep 1; Danio rerio; RNA Seq,GSM8369975 r1,GSM8369975,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_AMD_-010_cbc.fastq.gz,fastq,100371840.0,1672864.0,GSM8369975 r4,0:60,A:37032383;C:18500721;G:18308236;T:26522753;N:7747,60,,,,37032383,18500721,18308236,26522753,7747,SRX25158194,SRS21848816,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33099,SRR29654133,SRX25158193,SRS21848815,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,GSM8369974,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369974,GSM8369974: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369974 r1,GSM8369974,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_DMSO_-008_cbc.fastq.gz,fastq,161717520.0,2695292.0,GSM8369974 r1,0:60,A:58227035;C:29120167;G:33153372;T:41186362;N:30584,60,,,,58227035,29120167,33153372,41186362,30584,SRX25158193,SRS21848815,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33100,SRR29654134,SRX25158193,SRS21848815,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,GSM8369974,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369974,GSM8369974: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369974 r1,GSM8369974,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_DMSO_-008_cbc.fastq.gz,fastq,158810460.0,2646841.0,GSM8369974 r2,0:60,A:57059029;C:28475827;G:33090143;T:40160441;N:25020,60,,,,57059029,28475827,33090143,40160441,25020,SRX25158193,SRS21848815,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33101,SRR29654135,SRX25158193,SRS21848815,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,GSM8369974,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369974,GSM8369974: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369974 r1,GSM8369974,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_DMSO_-008_cbc.fastq.gz,fastq,164136600.0,2735610.0,GSM8369974 r3,0:60,A:59164760;C:29564845;G:33567677;T:41822336;N:16982,60,,,,59164760,29564845,33567677,41822336,16982,SRX25158193,SRS21848815,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33102,SRR29654136,SRX25158193,SRS21848815,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,GSM8369974,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369974,GSM8369974: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369974 r1,GSM8369974,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_DMSO_-008_cbc.fastq.gz,fastq,160939740.0,2682329.0,GSM8369974 r4,0:60,A:57905646;C:28872433;G:33402085;T:40746636;N:12940,60,,,,57905646,28872433,33402085,40746636,12940,SRX25158193,SRS21848815,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33103,SRR29654137,SRX25158192,SRS21848814,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,GSM8369973,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369973,GSM8369973: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369973 r1,GSM8369973,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_DMSO_-005_cbc.fastq.gz,fastq,294896760.0,4914946.0,GSM8369973 r1,0:60,A:113302890;C:53832811;G:54274745;T:73429183;N:57131,60,,,,113302890,53832811,54274745,73429183,57131,SRX25158192,SRS21848814,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33104,SRR29654138,SRX25158192,SRS21848814,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,GSM8369973,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369973,GSM8369973: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369973 r1,GSM8369973,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_DMSO_-005_cbc.fastq.gz,fastq,289658820.0,4827647.0,GSM8369973 r2,0:60,A:111138289;C:52646716;G:54190664;T:71637241;N:45910,60,,,,111138289,52646716,54190664,71637241,45910,SRX25158192,SRS21848814,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33105,SRR29654139,SRX25158192,SRS21848814,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,GSM8369973,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369973,GSM8369973: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369973 r1,GSM8369973,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_DMSO_-005_cbc.fastq.gz,fastq,299290800.0,4988180.0,GSM8369973 r3,0:60,A:115247052;C:54662821;G:54854607;T:74494831;N:31489,60,,,,115247052,54662821,54854607,74494831,31489,SRX25158192,SRS21848814,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33106,SRR29654140,SRX25158192,SRS21848814,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,GSM8369973,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369973,GSM8369973: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369973 r1,GSM8369973,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_DMSO_-005_cbc.fastq.gz,fastq,294091920.0,4901532.0,GSM8369973 r4,0:60,A:113032051;C:53520496;G:54754695;T:72762244;N:22434,60,,,,113032051,53520496,54754695,72762244,22434,SRX25158192,SRS21848814,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33107,SRR29654141,SRX25158191,SRS21848813,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,GSM8369972,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369972,GSM8369972: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369972 r1,GSM8369972,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_8hpi_DMSO_-004_cbc.fastq.gz,fastq,79542600.0,1325710.0,GSM8369972 r1,0:60,A:29052800;C:15106261;G:15344010;T:20023888;N:15641,60,,,,29052800,15106261,15344010,20023888,15641,SRX25158191,SRS21848813,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33108,SRR29654142,SRX25158191,SRS21848813,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,GSM8369972,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369972,GSM8369972: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369972 r1,GSM8369972,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_8hpi_DMSO_-004_cbc.fastq.gz,fastq,78150840.0,1302514.0,GSM8369972 r2,0:60,A:28490299;C:14795977;G:15298357;T:19554765;N:11442,60,,,,28490299,14795977,15298357,19554765,11442,SRX25158191,SRS21848813,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33109,SRR29654143,SRX25158191,SRS21848813,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,GSM8369972,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369972,GSM8369972: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369972 r1,GSM8369972,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_8hpi_DMSO_-004_cbc.fastq.gz,fastq,80719140.0,1345319.0,GSM8369972 r3,0:60,A:29548150;C:15350378;G:15486892;T:20325654;N:8066,60,,,,29548150,15350378,15486892,20325654,8066,SRX25158191,SRS21848813,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33110,SRR29654144,SRX25158191,SRS21848813,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,GSM8369972,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:8 xxx post injury DMSO,GSM8369972,GSM8369972: Neutrophils mpx+ 8 xxx post injury DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369972 r1,GSM8369972,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_8hpi_DMSO_-004_cbc.fastq.gz,fastq,79070760.0,1317846.0,GSM8369972 r4,0:60,A:28874779;C:14986197;G:15407453;T:19795922;N:6409,60,,,,28874779,14986197,15407453,19795922,6409,SRX25158191,SRS21848813,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33111,SRR29654145,SRX25158190,SRS21848812,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 3,GSM8369971,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369971,GSM8369971: Neutrophils mpx+ uninjured DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369971 r1,GSM8369971,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_DMSO_-009_cbc.fastq.gz,fastq,182391600.0,3039860.0,GSM8369971 r1,0:60,A:65842196;C:33929963;G:37226765;T:45358269;N:34407,60,,,,65842196,33929963,37226765,45358269,34407,SRX25158190,SRS21848812,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33112,SRR29654146,SRX25158190,SRS21848812,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 3,GSM8369971,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369971,GSM8369971: Neutrophils mpx+ uninjured DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369971 r1,GSM8369971,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_DMSO_-009_cbc.fastq.gz,fastq,179081640.0,2984694.0,GSM8369971 r2,0:60,A:64544585;C:33163305;G:37078756;T:44266486;N:28508,60,,,,64544585,33163305,37078756,44266486,28508,SRX25158190,SRS21848812,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33113,SRR29654147,SRX25158190,SRS21848812,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 3,GSM8369971,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369971,GSM8369971: Neutrophils mpx+ uninjured DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369971 r1,GSM8369971,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_DMSO_-009_cbc.fastq.gz,fastq,185068740.0,3084479.0,GSM8369971 r3,0:60,A:66919946;C:34450944;G:37652300;T:46025841;N:19709,60,,,,66919946,34450944,37652300,46025841,19709,SRX25158190,SRS21848812,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33114,SRR29654148,SRX25158190,SRS21848812,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 3,GSM8369971,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 3,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369971,GSM8369971: Neutrophils mpx+ uninjured DMSO biol rep 3; Danio rerio; RNA Seq,GSM8369971 r1,GSM8369971,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_DMSO_-009_cbc.fastq.gz,fastq,181507740.0,3025129.0,GSM8369971 r4,0:60,A:65513514;C:33675079;G:37432606;T:44872485;N:14056,60,,,,65513514,33675079,37432606,44872485,14056,SRX25158190,SRS21848812,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33115,SRR29654149,SRX25158189,SRS21848811,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 2,GSM8369970,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369970,GSM8369970: Neutrophils mpx+ uninjured DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369970 r1,GSM8369970,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_DMSO_-006_cbc.fastq.gz,fastq,45794760.0,763246.0,GSM8369970 r1,0:60,A:16928569;C:8492491;G:9006326;T:11358172;N:9202,60,,,,16928569,8492491,9006326,11358172,9202,SRX25158189,SRS21848811,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33116,SRR29654150,SRX25158189,SRS21848811,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 2,GSM8369970,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369970,GSM8369970: Neutrophils mpx+ uninjured DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369970 r1,GSM8369970,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_DMSO_-006_cbc.fastq.gz,fastq,45009960.0,750166.0,GSM8369970 r2,0:60,A:16608407;C:8309766;G:8990622;T:11094414;N:6751,60,,,,16608407,8309766,8990622,11094414,6751,SRX25158189,SRS21848811,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33117,SRR29654151,SRX25158189,SRS21848811,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 2,GSM8369970,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369970,GSM8369970: Neutrophils mpx+ uninjured DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369970 r1,GSM8369970,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_DMSO_-006_cbc.fastq.gz,fastq,46565880.0,776098.0,GSM8369970 r3,0:60,A:17217451;C:8640544;G:9146095;T:11557146;N:4644,60,,,,17217451,8640544,9146095,11557146,4644,SRX25158189,SRS21848811,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33118,SRR29654152,SRX25158189,SRS21848811,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 2,GSM8369970,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 2,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369970,GSM8369970: Neutrophils mpx+ uninjured DMSO biol rep 2; Danio rerio; RNA Seq,GSM8369970 r1,GSM8369970,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_DMSO_-006_cbc.fastq.gz,fastq,45624660.0,760411.0,GSM8369970 r4,0:60,A:16852187;C:8431444;G:9090234;T:11246907;N:3888,60,,,,16852187,8431444,9090234,11246907,3888,SRX25158189,SRS21848811,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33119,SRR29654153,SRX25158188,SRS21848810,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 1,GSM8369969,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369969,GSM8369969: Neutrophils mpx+ uninjured DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369969 r1,GSM8369969,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L001_Uninjured_DMSO_-003_cbc.fastq.gz,fastq,55867380.0,931123.0,GSM8369969 r1,0:60,A:20680346;C:10254613;G:10895370;T:14026951;N:10100,60,,,,20680346,10254613,10895370,14026951,10100,SRX25158188,SRS21848810,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33120,SRR29654154,SRX25158188,SRS21848810,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 1,GSM8369969,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369969,GSM8369969: Neutrophils mpx+ uninjured DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369969 r1,GSM8369969,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L002_Uninjured_DMSO_-003_cbc.fastq.gz,fastq,54889080.0,914818.0,GSM8369969 r2,0:60,A:20264110;C:10026239;G:10861715;T:13728039;N:8977,60,,,,20264110,10026239,10861715,13728039,8977,SRX25158188,SRS21848810,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33121,SRR29654155,SRX25158188,SRS21848810,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 1,GSM8369969,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369969,GSM8369969: Neutrophils mpx+ uninjured DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369969 r1,GSM8369969,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L003_Uninjured_DMSO_-003_cbc.fastq.gz,fastq,56764440.0,946074.0,GSM8369969 r3,0:60,A:21036899;C:10416984;G:11027830;T:14275499;N:7228,60,,,,21036899,10416984,11027830,14275499,7228,SRX25158188,SRS21848810,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 33122,SRR29654156,SRX25158188,SRS21848810,SRP517033,PRJNA1129880,Neutrophil immune profile guides spinal cord regeneration in zebrafish,GSE271113,Transcriptome Analysis,Spinal cord injury triggers a strong innate inflammatory response in both non regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet their role in the repair process particularly in a regenerative context remains largely unknown. Here we show that promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil specific RNA seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf a expression in macrophage/microglia populations. Conversely blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether these findings provide new insights into the role of neutrophils in spinal cord regeneration emphasizing the significant impact of their immune profile on the outcome of the repair process. Overall design: To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100 and DMSO treated samples. Grant ID: 2022.02766.PTDC Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective Funding agency: Fundaçåo para a Ciência e Tecnologia FCT Portugal,,pubmed:38925414,,Neutrophils mpx+ uninjured DMSO biol rep 1,GSM8369969,,tissue:Neutrophils mpx:GFP+|cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO|geo loc name:missing|collection date:missing,Neutrophils mpx+ uninjured DMSO biol rep 1,Libraries were paired end sequenced on a Nextseq 500 Illumina high output with a 1x75 bp Illumina Kit Read 1: 26 cycles index read: 6 cycles Read 2: 60 cycles. Read 1 was used to identify the Illumina library index and CEL Seq sample barcode and read 2 was aligned to the GRCz11 reference transcriptome using Burrow Wheeler aligner BWA MEM. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was done using the MapAndGo script. Normalization and differential gene expression analyses were conducted using DESeq2 v1.38 Assembly: GRCz11 zebrafish Supplementary files format and content: counts table generated using the MapAndGo script,Neutrophils mpx:GFP+,At 3 dpf larvae were anesthetized in 0.5 mM tricaine. Subsequenclty the spinal cord was fully transected at the level of the anal pore using the tip of a 30G needle. Next larvae were randomly distributed into different conditions and incubated with 25 uM AMD3100 supplemented with 0.1% DMSO from 4 xxx post injury hpi until 8 hpi.,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,Zebrafish embryos were raised at 28.5ºC in E3 media with Methylene blue,cell type:Neutrophils mpx:GFP+|treatment:uninjured DMSO,GSM8369969,GSM8369969: Neutrophils mpx+ uninjured DMSO biol rep 1; Danio rerio; RNA Seq,GSM8369969 r1,GSM8369969,1,Trunks containing the lesion site and same region of age matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV 15 mM HEPES 25 mM D Glucose 2% goat serum in HBSS without xxx and calcium incubated at 28.5ºC shaking at 300 rpm and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI 1ul/ml for 10 min at 4C. Single cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III BD Biosciences. All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500 4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL seq. Samples were barcoded with CEL seq primers during the reverse transcription and pooled post second strand synthesis TruSeq small RNA primers Illumina,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP517033,,,IMM-CT-b001_HW5F2BGXN_S6_L004_Uninjured_DMSO_-003_cbc.fastq.gz,fastq,55531080.0,925518.0,GSM8369969 r4,0:60,A:20538193;C:10150017;G:10949669;T:13888674;N:4527,60,,,,20538193,10150017,10949669,13888674,4527,SRX25158188,SRS21848810,SRA1913211,Instituto de Medicina Molecular,Instituto de Medicina Molecular,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Portugal,2024-06-29,Larval,Larval,Blood,Hematopoietic System 38073,SRR1531483,SRX665263,SRS670077,SRP045064,PRJNA256975,Genome wide RNA tomography in the zebrafish embryo,GSE59873,Other,Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes they are unable to yield spatially resolved information in embryos or tissues. Microscopy based approaches using for example in situ hybridization can provide spatial information about gene expression but are limited to analyzing one or a few genes at a time. Here we present a method where we combine traditional histological techniques with low input RNA sequencing and mathematical image reconstruction to generate a high resolution genome wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly our method enables searching for genes that are expressed in specific spatial patterns without xxx image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. Overall design: To generate spatially resolved RNA seq data for zebrafish embryos shield stage 10 somites 15 somites 18 somites and mouse forelimbs E10.5 we cryosectioned samples extracted RNA from the individual sections and amplified and barcoded mRNA using the CEL seq protocol Hashimshony et al. Cell Reports 2012 with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired end to improve three prime annotations.,,pubmed:25417113,,18 somites stage straightened secti1d tail to head,GSM1448820,,tissue:deyolked embryo head removed|Stage:18 somites|sectioning direction:tail to head|section thickness:24 µm|embedding strategy:deyolked and straightened embryo,18 somites stage straightened secti1d tail to head,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The mouse transcriptome contained all RefSeq gene models based on the mouse genome release mm10. The zebrafish transcriptome was based on genome release zv9 and contained improved gene annotations as described in the manuscript Table S1. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 Genome build: mm10 and zv9 with improved three prime annotation Supplementary files format and content: text files: read count matrices in which rows correspond to genes and columns to sections,deyolked embryo head removed,Unfixed samples zebrafish embryos or mouse forelimbs were embedded in tissue freezing medium. Blocks were cryosectioned and individual slices were transferred to Eppendorf tubes on dry ice.,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,,Stage:18 somites|sectioning direction:tail to head|section thickness:24 µm|embedding strategy:deyolked and straightened embryo,GSM1448820,GSM1448820: 18 somites stage straightened secti1d tail to head; Danio rerio; RNA Seq,GSM1448820,,1,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,GEO Accession:GSM1448820,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP045064,,,dr_18ss_TH_R2.fastq.gz dr_18ss_TH_R1.fastq.gz,fastq fastq,15779230884.0,154698342.0,GSM1448820 r1,0:51 1:51,A:3451000604;C:2392800593;G:2522666152;T:7412137093;N:626442,51,51,,,3451000604,2392800593,2522666152,7412137093,626442,SRX665263,SRS670077,SRA177126,GEO,Hubrecht Institute,2,0.11555,0.70031,0.08647,0.12212,0.98238,0.78946,0.48905,0.5514,51,51,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,sc,single_cell_plate,celseq,,Netherlands,2014-07-29,Segmentation,Embryo,Embryo Imprecise,All anatomical structures 38074,SRR1531482,SRX665262,SRS670076,SRP045064,PRJNA256975,Genome wide RNA tomography in the zebrafish embryo,GSE59873,Other,Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes they are unable to yield spatially resolved information in embryos or tissues. Microscopy based approaches using for example in situ hybridization can provide spatial information about gene expression but are limited to analyzing one or a few genes at a time. Here we present a method where we combine traditional histological techniques with low input RNA sequencing and mathematical image reconstruction to generate a high resolution genome wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly our method enables searching for genes that are expressed in specific spatial patterns without xxx image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. Overall design: To generate spatially resolved RNA seq data for zebrafish embryos shield stage 10 somites 15 somites 18 somites and mouse forelimbs E10.5 we cryosectioned samples extracted RNA from the individual sections and amplified and barcoded mRNA using the CEL seq protocol Hashimshony et al. Cell Reports 2012 with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired end to improve three prime annotations.,,pubmed:25417113,,15 somites stage straightened secti1d tail to head replicate,GSM1448819,,tissue:deyolked embryo|Stage:15 somites|sectioning direction:tail to head|section thickness:18 µm|embedding strategy:deyolked and straightened embryo,15 somites stage straightened secti1d tail to head replicate,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The mouse transcriptome contained all RefSeq gene models based on the mouse genome release mm10. The zebrafish transcriptome was based on genome release zv9 and contained improved gene annotations as described in the manuscript Table S1. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 Genome build: mm10 and zv9 with improved three prime annotation Supplementary files format and content: text files: read count matrices in which rows correspond to genes and columns to sections,deyolked embryo,Unfixed samples zebrafish embryos or mouse forelimbs were embedded in tissue freezing medium. Blocks were cryosectioned and individual slices were transferred to Eppendorf tubes on dry ice.,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,,Stage:15 somites|sectioning direction:tail to head|section thickness:18 µm|embedding strategy:deyolked and straightened embryo,GSM1448819,GSM1448819: 15 somites stage straightened secti1d tail to head replicate; Danio rerio; RNA Seq,GSM1448819,,1,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,GEO Accession:GSM1448819,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP045064,,,dr_15ss_TH2_R2.fastq.gz dr_15ss_TH2_R1.fastq.gz,fastq fastq,4502603442.0,44143171.0,GSM1448819 r1,0:51 1:51,A:1010801791;C:688133305;G:712006267;T:2047960758;N:43701321,51,51,,,1010801791,688133305,712006267,2047960758,43701321,SRX665262,SRS670076,SRA177126,GEO,Hubrecht Institute,2,0.0903,0.78618,0.08054,0.12953,0.98703,0.78046,0.53574,0.52775,51,51,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,sc,single_cell_plate,celseq,,Netherlands,2014-07-29,Segmentation,Embryo,Embryo Imprecise,All anatomical structures 38075,SRR1531481,SRX665261,SRS670075,SRP045064,PRJNA256975,Genome wide RNA tomography in the zebrafish embryo,GSE59873,Other,Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes they are unable to yield spatially resolved information in embryos or tissues. Microscopy based approaches using for example in situ hybridization can provide spatial information about gene expression but are limited to analyzing one or a few genes at a time. Here we present a method where we combine traditional histological techniques with low input RNA sequencing and mathematical image reconstruction to generate a high resolution genome wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly our method enables searching for genes that are expressed in specific spatial patterns without xxx image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. Overall design: To generate spatially resolved RNA seq data for zebrafish embryos shield stage 10 somites 15 somites 18 somites and mouse forelimbs E10.5 we cryosectioned samples extracted RNA from the individual sections and amplified and barcoded mRNA using the CEL seq protocol Hashimshony et al. Cell Reports 2012 with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired end to improve three prime annotations.,,pubmed:25417113,,sections 97 108 for samples dr 15ss TH and dr 15ss HT,GSM1448818,,tissue:deyolked embryo|Stage:15 somites|sectioning direction:head to tail and tail to head|section thickness:18 µm|embedding strategy:deyolked and straightened embryo,sections 97 108 for samples dr 15ss TH and dr 15ss HT,Paired end reads were aligned to the transcriptome using bwa version 0.6.2 with default parameters. The mouse transcriptome contained all RefSeq gene models based on the mouse genome release mm10. The zebrafish transcriptome was based on genome release zv9 and contained improved gene annotations as described in the manuscript Table S1. The right mate of each read pair was mapped to the ensemble of all transcripts and to the set of 92 ERCC spike ins in sense direction. Reads mapping equally to multiple loci were discarded. Mapped reads were assigned to sections based on barcodes according to the CEL seq protocol Hashimshony et al. Cell Reports 2012 Genome build: mm10 and zv9 with improved three prime annotation Supplementary files format and content: text files: read count matrices in which rows correspond to genes and columns to sections,deyolked embryo,Unfixed samples zebrafish embryos or mouse forelimbs were embedded in tissue freezing medium. Blocks were cryosectioned and individual slices were transferred to Eppendorf tubes on dry ice.,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,,Stage:15 somites|sectioning direction:head to tail and tail to head|section thickness:18 µm|embedding strategy:deyolked and straightened embryo,GSM1448818,GSM1448818: sections 97 108 for samples dr 15ss TH and dr 15ss HT; Danio rerio; RNA Seq,GSM1448818,,1,RNA was isolated by TRIzol extraction mRNA was barcoded and amplified using the CEL seq protocol Hashimshony et al. Cell Reports 2012,GEO Accession:GSM1448818,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP045064,,,dr_15ss_TH97-108_HT97-108_R2.fastq.gz dr_15ss_TH97-108_HT97-108_R1.fastq.gz,fastq fastq,472071606.0,4628153.0,GSM1448818 r1,0:51 1:51,A:108013079;C:76852222;G:73612321;T:208300498;N:5293486,51,51,,,108013079,76852222,73612321,208300498,5293486,SRX665261,SRS670075,SRA177126,GEO,Hubrecht Institute,2,0.06997,0.71056,0.06103,0.12966,0.9865,0.79446,0.53118,0.53673,51,51,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,sc,single_cell_plate,celseq,,Netherlands,2014-07-29,Segmentation,Embryo,Embryo Imprecise,All anatomical structures