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 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 38122,SRR1555598,SRX684706,SRS687823,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0106,GSM1483841,,tissue:single embryo,Metazome ZF timecourse sample 0106,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4240,GSM1483841,GSM1483841: Metazome ZF timecourse sample 0106; Danio rerio; RNA Seq,GSM1483841,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483841,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0106.fastq.gz,fastq,3911705.0,111763.0,GSM1483841 r1,0:35,A:1100064;C:790487;G:836206;T:1169251;N:15697,35,,,,1100064,790487,836206,1169251,15697,SRX684706,SRS687823,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.72414,,0.09461,,0.96725,,0.4942,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38123,SRR1555597,SRX684705,SRS687821,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0105,GSM1483840,,tissue:single embryo,Metazome ZF timecourse sample 0105,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4200,GSM1483840,GSM1483840: Metazome ZF timecourse sample 0105; Danio rerio; RNA Seq,GSM1483840,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483840,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0105.fastq.gz,fastq,32877145.0,939347.0,GSM1483840 r1,0:35,A:9524894;C:6625944;G:6696548;T:9953576;N:76183,35,,,,9524894,6625944,6696548,9953576,76183,SRX684705,SRS687821,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.74436,,0.07095,,0.88201,,0.47612,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38124,SRR1555596,SRX684704,SRS687822,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0104,GSM1483839,,tissue:single embryo,Metazome ZF timecourse sample 0104,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4160,GSM1483839,GSM1483839: Metazome ZF timecourse sample 0104; Danio rerio; RNA Seq,GSM1483839,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483839,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0104.fastq.gz,fastq,83810020.0,2394572.0,GSM1483839 r1,0:35,A:24075610;C:16799998;G:16694424;T:25967423;N:272565,35,,,,24075610,16799998,16694424,25967423,272565,SRX684704,SRS687822,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.7588,,0.07993,,0.84449,,0.49785,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38125,SRR1555595,SRX684703,SRS687820,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0103,GSM1483838,,tissue:single embryo,Metazome ZF timecourse sample 0103,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4120,GSM1483838,GSM1483838: Metazome ZF timecourse sample 0103; Danio rerio; RNA Seq,GSM1483838,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483838,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0103.fastq.gz,fastq,98206150.0,2805890.0,GSM1483838 r1,0:35,A:28361916;C:19232789;G:19589483;T:30798968;N:222994,35,,,,28361916,19232789,19589483,30798968,222994,SRX684703,SRS687820,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75509,,0.13429,,0.81117,,0.49102,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38126,SRR1555594,SRX684702,SRS687819,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0102,GSM1483837,,tissue:single embryo,Metazome ZF timecourse sample 0102,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4080,GSM1483837,GSM1483837: Metazome ZF timecourse sample 0102; Danio rerio; RNA Seq,GSM1483837,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483837,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0102.fastq.gz,fastq,192817835.0,5509081.0,GSM1483837 r1,0:35,A:55969077;C:36880901;G:37835921;T:61101558;N:1030378,35,,,,55969077,36880901,37835921,61101558,1030378,SRX684702,SRS687819,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.73387,,0.17245,,0.80164,,0.48657,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38127,SRR1555593,SRX684701,SRS687818,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0101,GSM1483836,,tissue:single embryo,Metazome ZF timecourse sample 0101,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4040,GSM1483836,GSM1483836: Metazome ZF timecourse sample 0101; Danio rerio; RNA Seq,GSM1483836,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483836,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0101.fastq.gz,fastq,374339455.0,10695413.0,GSM1483836 r1,0:35,A:112191846;C:73679996;G:73185024;T:114766825;N:515764,35,,,,112191846,73679996,73185024,114766825,515764,SRX684701,SRS687818,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.83131,,0.16618,,0.77812,,0.49895,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38128,SRR1555592,SRX684700,SRS687816,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0100,GSM1483835,,tissue:single embryo,Metazome ZF timecourse sample 0100,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:4000,GSM1483835,GSM1483835: Metazome ZF timecourse sample 0100; Danio rerio; RNA Seq,GSM1483835,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483835,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0100.fastq.gz,fastq,15239420.0,435412.0,GSM1483835 r1,0:35,A:4361241;C:3088134;G:3269084;T:4498093;N:22868,35,,,,4361241,3088134,3269084,4498093,22868,SRX684700,SRS687816,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.7702,,0.17879,,0.92904,,0.505,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38129,SRR1555591,SRX684699,SRS687817,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0099,GSM1483834,,tissue:single embryo,Metazome ZF timecourse sample 0099,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3960,GSM1483834,GSM1483834: Metazome ZF timecourse sample 0099; Danio rerio; RNA Seq,GSM1483834,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483834,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0099.fastq.gz,fastq,284196500.0,8119900.0,GSM1483834 r1,0:35,A:81991733;C:57440756;G:58362818;T:86010858;N:390335,35,,,,81991733,57440756,58362818,86010858,390335,SRX684699,SRS687817,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.81284,,0.1715,,0.78468,,0.49472,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38130,SRR1555590,SRX684698,SRS687815,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0098,GSM1483833,,tissue:single embryo,Metazome ZF timecourse sample 0098,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3920,GSM1483833,GSM1483833: Metazome ZF timecourse sample 0098; Danio rerio; RNA Seq,GSM1483833,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483833,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0098.fastq.gz,fastq,244245015.0,6978429.0,GSM1483833 r1,0:35,A:70141725;C:48500061;G:50748992;T:74478239;N:375998,35,,,,70141725,48500061,50748992,74478239,375998,SRX684698,SRS687815,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.79668,,0.17718,,0.79271,,0.45966,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38131,SRR1555589,SRX684697,SRS687814,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0097,GSM1483832,,tissue:single embryo,Metazome ZF timecourse sample 0097,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3880,GSM1483832,GSM1483832: Metazome ZF timecourse sample 0097; Danio rerio; RNA Seq,GSM1483832,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483832,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0097.fastq.gz,fastq,215347510.0,6152786.0,GSM1483832 r1,0:35,A:62928172;C:42420829;G:43902946;T:65803342;N:292221,35,,,,62928172,42420829,43902946,65803342,292221,SRX684697,SRS687814,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80605,,0.17396,,0.78873,,0.50241,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38132,SRR1555588,SRX684696,SRS687813,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0096,GSM1483831,,tissue:single embryo,Metazome ZF timecourse sample 0096,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3840,GSM1483831,GSM1483831: Metazome ZF timecourse sample 0096; Danio rerio; RNA Seq,GSM1483831,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483831,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0096.fastq.gz,fastq,13992230.0,399778.0,GSM1483831 r1,0:35,A:4077402;C:2837417;G:2856511;T:4200750;N:20150,35,,,,4077402,2837417,2856511,4200750,20150,SRX684696,SRS687813,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.77543,,0.14778,,0.93288,,0.48967,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38133,SRR1555587,SRX684695,SRS687811,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0095,GSM1483830,,tissue:single embryo,Metazome ZF timecourse sample 0095,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3800,GSM1483830,GSM1483830: Metazome ZF timecourse sample 0095; Danio rerio; RNA Seq,GSM1483830,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483830,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0095.fastq.gz,fastq,290151435.0,8290041.0,GSM1483830 r1,0:35,A:86694666;C:56953853;G:57315219;T:88811589;N:376108,35,,,,86694666,56953853,57315219,88811589,376108,SRX684695,SRS687811,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.79098,,0.18155,,0.79034,,0.51338,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38134,SRR1555586,SRX684694,SRS687810,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0094,GSM1483829,,tissue:single embryo,Metazome ZF timecourse sample 0094,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3760,GSM1483829,GSM1483829: Metazome ZF timecourse sample 0094; Danio rerio; RNA Seq,GSM1483829,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483829,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0094.fastq.gz,fastq,645422925.0,18440655.0,GSM1483829 r1,0:35,A:186408265;C:127218686;G:131406509;T:199399112;N:990353,35,,,,186408265,127218686,131406509,199399112,990353,SRX684694,SRS687810,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.81184,,0.15572,,0.78496,,0.48733,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38135,SRR1555585,SRX684693,SRS687812,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0093,GSM1483828,,tissue:single embryo,Metazome ZF timecourse sample 0093,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3720,GSM1483828,GSM1483828: Metazome ZF timecourse sample 0093; Danio rerio; RNA Seq,GSM1483828,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483828,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0093.fastq.gz,fastq,82574205.0,2359263.0,GSM1483828 r1,0:35,A:24239984;C:16625180;G:16309055;T:25123568;N:276418,35,,,,24239984,16625180,16309055,25123568,276418,SRX684693,SRS687812,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75557,,0.14554,,0.82205,,0.49736,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38136,SRR1555584,SRX684692,SRS687809,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0092,GSM1483827,,tissue:single embryo,Metazome ZF timecourse sample 0092,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3680,GSM1483827,GSM1483827: Metazome ZF timecourse sample 0092; Danio rerio; RNA Seq,GSM1483827,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483827,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0092.fastq.gz,fastq,72186905.0,2062483.0,GSM1483827 r1,0:35,A:21349438;C:14361391;G:14472348;T:21766197;N:237531,35,,,,21349438,14361391,14472348,21766197,237531,SRX684692,SRS687809,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.74348,,0.13614,,0.8309,,0.51328,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38137,SRR1555583,SRX684691,SRS687808,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0091,GSM1483826,,tissue:single embryo,Metazome ZF timecourse sample 0091,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3640,GSM1483826,GSM1483826: Metazome ZF timecourse sample 0091; Danio rerio; RNA Seq,GSM1483826,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483826,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0091.fastq.gz,fastq,18405940.0,525884.0,GSM1483826 r1,0:35,A:5374937;C:3766785;G:3774551;T:5451369;N:38298,35,,,,5374937,3766785,3774551,5451369,38298,SRX684691,SRS687808,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.74005,,0.11089,,0.91494,,0.49823,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38138,SRR1555582,SRX684690,SRS687807,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0090,GSM1483825,,tissue:single embryo,Metazome ZF timecourse sample 0090,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3600,GSM1483825,GSM1483825: Metazome ZF timecourse sample 0090; Danio rerio; RNA Seq,GSM1483825,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483825,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0090.fastq.gz,fastq,141938265.0,4055379.0,GSM1483825 r1,0:35,A:41371650;C:28115945;G:29356068;T:42485478;N:609124,35,,,,41371650,28115945,29356068,42485478,609124,SRX684690,SRS687807,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.71151,,0.17711,,0.82789,,0.52369,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38139,SRR1555581,SRX684689,SRS687806,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0089,GSM1483824,,tissue:single embryo,Metazome ZF timecourse sample 0089,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3560,GSM1483824,GSM1483824: Metazome ZF timecourse sample 0089; Danio rerio; RNA Seq,GSM1483824,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483824,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0089.fastq.gz,fastq,99569015.0,2844829.0,GSM1483824 r1,0:35,A:28763571;C:20065085;G:19332839;T:31088193;N:319327,35,,,,28763571,20065085,19332839,31088193,319327,SRX684689,SRS687806,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.74177,,0.13103,,0.81854,,0.51184,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38140,SRR1555580,SRX684688,SRS687805,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0088,GSM1483823,,tissue:single embryo,Metazome ZF timecourse sample 0088,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3520,GSM1483823,GSM1483823: Metazome ZF timecourse sample 0088; Danio rerio; RNA Seq,GSM1483823,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483823,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0088.fastq.gz,fastq,168790055.0,4822573.0,GSM1483823 r1,0:35,A:49034449;C:32785565;G:33439918;T:52997820;N:532303,35,,,,49034449,32785565,33439918,52997820,532303,SRX684688,SRS687805,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.73454,,0.15775,,0.8047,,0.50587,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38141,SRR1555579,SRX684687,SRS687803,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0087,GSM1483822,,tissue:single embryo,Metazome ZF timecourse sample 0087,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3480,GSM1483822,GSM1483822: Metazome ZF timecourse sample 0087; Danio rerio; RNA Seq,GSM1483822,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483822,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0087.fastq.gz,fastq,83155975.0,2375885.0,GSM1483822 r1,0:35,A:23985678;C:16871573;G:17048421;T:25086100;N:164203,35,,,,23985678,16871573,17048421,25086100,164203,SRX684687,SRS687803,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75055,,0.10258,,0.83952,,0.49914,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38142,SRR1555578,SRX684686,SRS687804,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0086,GSM1483821,,tissue:single embryo,Metazome ZF timecourse sample 0086,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3440,GSM1483821,GSM1483821: Metazome ZF timecourse sample 0086; Danio rerio; RNA Seq,GSM1483821,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483821,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0086.fastq.gz,fastq,36772155.0,1050633.0,GSM1483821 r1,0:35,A:10536765;C:7377251;G:7701105;T:11014088;N:142946,35,,,,10536765,7377251,7701105,11014088,142946,SRX684686,SRS687804,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.745,,0.08312,,0.88209,,0.52632,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38143,SRR1555577,SRX684685,SRS687802,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0085,GSM1483820,,tissue:single embryo,Metazome ZF timecourse sample 0085,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3400,GSM1483820,GSM1483820: Metazome ZF timecourse sample 0085; Danio rerio; RNA Seq,GSM1483820,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483820,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0085.fastq.gz,fastq,142281650.0,4065190.0,GSM1483820 r1,0:35,A:41238155;C:28228882;G:28727190;T:43901346;N:186077,35,,,,41238155,28228882,28727190,43901346,186077,SRX684685,SRS687802,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80695,,0.14045,,0.80361,,0.5,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38144,SRR1555576,SRX684684,SRS687800,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0084,GSM1483819,,tissue:single embryo,Metazome ZF timecourse sample 0084,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3360,GSM1483819,GSM1483819: Metazome ZF timecourse sample 0084; Danio rerio; RNA Seq,GSM1483819,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483819,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0084.fastq.gz,fastq,147589295.0,4216837.0,GSM1483819 r1,0:35,A:42100013;C:30125860;G:31767504;T:43391728;N:204190,35,,,,42100013,30125860,31767504,43391728,204190,SRX684684,SRS687800,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80373,,0.08661,,0.83157,,0.51923,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38145,SRR1555575,SRX684683,SRS687801,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0083,GSM1483818,,tissue:single embryo,Metazome ZF timecourse sample 0083,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3320,GSM1483818,GSM1483818: Metazome ZF timecourse sample 0083; Danio rerio; RNA Seq,GSM1483818,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483818,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0083.fastq.gz,fastq,147243985.0,4206971.0,GSM1483818 r1,0:35,A:42533757;C:29874233;G:30500611;T:44132167;N:203217,35,,,,42533757,29874233,30500611,44132167,203217,SRX684683,SRS687801,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80738,,0.08642,,0.82219,,0.49396,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38146,SRR1555574,SRX684682,SRS687799,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0082,GSM1483817,,tissue:single embryo,Metazome ZF timecourse sample 0082,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3280,GSM1483817,GSM1483817: Metazome ZF timecourse sample 0082; Danio rerio; RNA Seq,GSM1483817,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483817,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0082.fastq.gz,fastq,96747735.0,2764221.0,GSM1483817 r1,0:35,A:26768730;C:19578598;G:21713242;T:28549716;N:137449,35,,,,26768730,19578598,21713242,28549716,137449,SRX684682,SRS687799,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80738,,0.09417,,0.85167,,0.50713,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38147,SRR1555573,SRX684681,SRS687798,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0081,GSM1483816,,tissue:single embryo,Metazome ZF timecourse sample 0081,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3240,GSM1483816,GSM1483816: Metazome ZF timecourse sample 0081; Danio rerio; RNA Seq,GSM1483816,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483816,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0081.fastq.gz,fastq,158402895.0,4525797.0,GSM1483816 r1,0:35,A:46939694;C:31610300;G:32050592;T:47571349;N:230960,35,,,,46939694,31610300,32050592,47571349,230960,SRX684681,SRS687798,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.78709,,0.11916,,0.82351,,0.48714,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38148,SRR1555572,SRX684680,SRS687797,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0080,GSM1483815,,tissue:single embryo,Metazome ZF timecourse sample 0080,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3200,GSM1483815,GSM1483815: Metazome ZF timecourse sample 0080; Danio rerio; RNA Seq,GSM1483815,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483815,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0080.fastq.gz,fastq,1432375.0,40925.0,GSM1483815 r1,0:35,A:422951;C:297837;G:308257;T:400778;N:2552,35,,,,422951,297837,308257,400778,2552,SRX684680,SRS687797,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.7103,,0.12471,,0.9811,,0.57619,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38149,SRR1555571,SRX684679,SRS687796,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0079,GSM1483814,,tissue:single embryo,Metazome ZF timecourse sample 0079,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3160,GSM1483814,GSM1483814: Metazome ZF timecourse sample 0079; Danio rerio; RNA Seq,GSM1483814,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483814,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0079.fastq.gz,fastq,171937640.0,4912504.0,GSM1483814 r1,0:35,A:49235964;C:34067309;G:35846095;T:52553397;N:234875,35,,,,49235964,34067309,35846095,52553397,234875,SRX684679,SRS687796,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.79389,,0.14252,,0.81107,,0.50474,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38150,SRR1555570,SRX684678,SRS687795,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0078,GSM1483813,,tissue:single embryo,Metazome ZF timecourse sample 0078,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3120,GSM1483813,GSM1483813: Metazome ZF timecourse sample 0078; Danio rerio; RNA Seq,GSM1483813,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483813,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0078.fastq.gz,fastq,220246950.0,6292770.0,GSM1483813 r1,0:35,A:63723749;C:43666354;G:45504302;T:67009596;N:342949,35,,,,63723749,43666354,45504302,67009596,342949,SRX684678,SRS687795,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.80813,,0.1512,,0.80174,,0.49937,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38151,SRR1555569,SRX684677,SRS687794,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0077,GSM1483812,,tissue:single embryo,Metazome ZF timecourse sample 0077,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3080,GSM1483812,GSM1483812: Metazome ZF timecourse sample 0077; Danio rerio; RNA Seq,GSM1483812,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483812,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0077.fastq.gz,fastq,93138675.0,2661105.0,GSM1483812 r1,0:35,A:27102533;C:18314712;G:18498218;T:29006764;N:216448,35,,,,27102533,18314712,18498218,29006764,216448,SRX684677,SRS687794,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75818,,0.11499,,0.81572,,0.50169,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38152,SRR1555568,SRX684676,SRS687793,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0076,GSM1483811,,tissue:single embryo,Metazome ZF timecourse sample 0076,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3040,GSM1483811,GSM1483811: Metazome ZF timecourse sample 0076; Danio rerio; RNA Seq,GSM1483811,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483811,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0076.fastq.gz,fastq,118512415.0,3386069.0,GSM1483811 r1,0:35,A:33806370;C:23395934;G:24547522;T:36374942;N:387647,35,,,,33806370,23395934,24547522,36374942,387647,SRX684676,SRS687793,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75326,,0.1088,,0.81667,,0.49341,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38153,SRR1555567,SRX684675,SRS687792,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0075,GSM1483810,,tissue:single embryo,Metazome ZF timecourse sample 0075,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:3000,GSM1483810,GSM1483810: Metazome ZF timecourse sample 0075; Danio rerio; RNA Seq,GSM1483810,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483810,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0075.fastq.gz,fastq,2520525.0,72015.0,GSM1483810 r1,0:35,A:818327;C:420638;G:441967;T:833315;N:6278,35,,,,818327,420638,441967,833315,6278,SRX684675,SRS687792,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.7119,,0.22445,,0.97368,,0.51561,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38154,SRR1555566,SRX684674,SRS687791,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0074,GSM1483809,,tissue:single embryo,Metazome ZF timecourse sample 0074,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2960,GSM1483809,GSM1483809: Metazome ZF timecourse sample 0074; Danio rerio; RNA Seq,GSM1483809,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483809,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0074.fastq.gz,fastq,95385710.0,2725306.0,GSM1483809 r1,0:35,A:27032549;C:18681500;G:19490852;T:29708978;N:471831,35,,,,27032549,18681500,19490852,29708978,471831,SRX684674,SRS687791,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.75216,,0.09004,,0.83469,,0.48768,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38155,SRR1555565,SRX684673,SRS687790,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0073,GSM1483808,,tissue:single embryo,Metazome ZF timecourse sample 0073,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2920,GSM1483808,GSM1483808: Metazome ZF timecourse sample 0073; Danio rerio; RNA Seq,GSM1483808,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483808,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0073.fastq.gz,fastq,149165835.0,4261881.0,GSM1483808 r1,0:35,A:42991931;C:29913953;G:30117935;T:45803552;N:338464,35,,,,42991931,29913953,30117935,45803552,338464,SRX684673,SRS687790,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.76488,,0.10545,,0.80586,,0.5046,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38156,SRR1555564,SRX684672,SRS687789,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0072,GSM1483807,,tissue:single embryo,Metazome ZF timecourse sample 0072,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2880,GSM1483807,GSM1483807: Metazome ZF timecourse sample 0072; Danio rerio; RNA Seq,GSM1483807,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483807,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0072.fastq.gz,fastq,203829675.0,5823705.0,GSM1483807 r1,0:35,A:61171233;C:40047517;G:44515900;T:57461403;N:633622,35,,,,61171233,40047517,44515900,57461403,633622,SRX684672,SRS687789,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.65225,,0.10254,,0.82014,,0.50437,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38157,SRR1555563,SRX684671,SRS687788,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0071,GSM1483806,,tissue:single embryo,Metazome ZF timecourse sample 0071,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2840,GSM1483806,GSM1483806: Metazome ZF timecourse sample 0071; Danio rerio; RNA Seq,GSM1483806,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483806,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0071.fastq.gz,fastq,107894360.0,3082696.0,GSM1483806 r1,0:35,A:30475809;C:22092085;G:22685204;T:32400616;N:240646,35,,,,30475809,22092085,22685204,32400616,240646,SRX684671,SRS687788,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.77637,,0.10375,,0.81574,,0.50918,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38158,SRR1555562,SRX684670,SRS687787,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0070,GSM1483805,,tissue:single embryo,Metazome ZF timecourse sample 0070,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2800,GSM1483805,GSM1483805: Metazome ZF timecourse sample 0070; Danio rerio; RNA Seq,GSM1483805,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483805,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0070.fastq.gz,fastq,102775820.0,2936452.0,GSM1483805 r1,0:35,A:29014067;C:20865233;G:21656956;T:30738746;N:500818,35,,,,29014067,20865233,21656956,30738746,500818,SRX684670,SRS687787,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.7643,,0.09535,,0.8325,,0.51201,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38159,SRR1555561,SRX684669,SRS687786,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0069,GSM1483804,,tissue:single embryo,Metazome ZF timecourse sample 0069,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2760,GSM1483804,GSM1483804: Metazome ZF timecourse sample 0069; Danio rerio; RNA Seq,GSM1483804,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483804,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0069.fastq.gz,fastq,178882445.0,5110927.0,GSM1483804 r1,0:35,A:51610621;C:35847602;G:36898756;T:54275519;N:249947,35,,,,51610621,35847602,36898756,54275519,249947,SRX684669,SRS687786,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.81481,,0.1212,,0.80338,,0.48647,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38160,SRR1555560,SRX684668,SRS687784,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0068,GSM1483803,,tissue:single embryo,Metazome ZF timecourse sample 0068,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2720,GSM1483803,GSM1483803: Metazome ZF timecourse sample 0068; Danio rerio; RNA Seq,GSM1483803,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483803,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0068.fastq.gz,fastq,289924775.0,8283565.0,GSM1483803 r1,0:35,A:85968202;C:57218562;G:58533597;T:87797178;N:407236,35,,,,85968202,57218562,58533597,87797178,407236,SRX684668,SRS687784,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.78411,,0.12015,,0.80736,,0.5018,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures 38161,SRR1555559,SRX684667,SRS687785,SRP045679,PRJNA258656,Danio rerio high resolution developmental transcriptomic time course,GSE60619,Transcriptome Analysis,Classical embryological studies revealed that during mid embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum including a segmented worm a flatworm a roundworm a water bear a fruitfly a sea urchin a zebrafish a sea anemone a sponge and a comb jelly. For each species we collected the embryonic transcriptomes at 100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. Overall design: 106 single embryo samples,parent bioproject:PRJNA287810,pubmed:26886793,,Metazome ZF timecourse sample 0067,GSM1483802,,tissue:single embryo,Metazome ZF timecourse sample 0067,Libraries were sequenced on the Illumina HiSeq2000 according to standard paired end sequencing protocols. Primary analysis done in RTA 1.17.20 1.13.48 Conversion from BCL to FASTQ and deumltiplexing using CASAVA 1.8 configureBclToFastq.pl fastq cluster count 1234567890 mismatches 0 use bases mask Y15n I6n Y35n Filter and read trimming barcode minimum quality of 10. trimming of read2 to 35 bases not required. CEL Seq Hashimshony et a. 2012l demultiplexing of second mate using first mate barcode allowing no mismatches in barcode. bowtie2 version 2.1.0 against Zv9 genome Read counting with htseq count version 0.5.4p3. Using on Ensemble Zv9 annotations Genome build: Zv9 Supplementary files format and content: tabular Expression matrix,single embryo,,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,zebrafish fertilization was performed in the lab of Karina Yaniv Weizmann Institute Israel. Four female and 1 male Danio rerio fish were mixed in a breeder tank and encouraged to spawn by tilting the tank and using artificial trees. post females started to release their eggs males quickly released sperm and fertilized eggs were collected into Zebrafish embryo medium as described in REF. Fertilized eggs were sampled in a small volume of medium every 40 minutes from fertilization into the cap of an 1.5 ml Eppendorf tube. Excess water was removed using a micro mouthpipette and the embryo flash frozen in liquid nitrogen.,time min post fertilization:2680,GSM1483802,GSM1483802: Metazome ZF timecourse sample 0067; Danio rerio; RNA Seq,GSM1483802,,1,RNA was isolated using TRIzol as previously described Levin et al. 2012. The CEL Seq protocol Hashimshony et al. 2012 was used to amplify and sequence. CEL seq multiplexing barocdes were used.,GEO Accession:GSM1483802,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP045679,,,Metazome_ZF_timecourse_sample_0067.fastq.gz,fastq,12717355.0,363353.0,GSM1483802 r1,0:35,A:3757539;C:2504848;G:2608725;T:3830804;N:15439,35,,,,3757539,2504848,2608725,3830804,15439,SRX684667,SRS687785,SRA179995,GEO,"Yanai, Biology, Technion - Israel Institute of Technology",1,0.77835,,0.12605,,0.92604,,0.45926,,35,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,celseq,,Israel,2014-08-21,Zygote,Embryo,Embryo Imprecise,All anatomical structures