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
29808,SRR27450807,SRX23122487,SRS20076037,SRP482328,PRJNA1061565,Akt is the main kinase mediating the embryonic specification of artery cells,GSE252648,Transcriptome Analysis,This study aimed at understanding the function of Akt signaling in vascular development in Zebrafish. Overall design: This study used Zebrafish endothelial cells sorted from WT and akt full mutant embryos at 24hpf. Endothelial cells were sorted via the use of transgenic line kdrl:mCherry+. Only mCherry+ were sequenced. So here you can find sample WT and sample akt mutant.,,pubmed:39101673,,Zebrafish  EC  24hpf  Aktmutant,GSM8004755,,source name:FAC sorted cells|tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:Aktmutant|geo loc name:missing|collection date:missing,Zebrafish  EC  24hpf  Aktmutant,The barcoded processing  gene counting and aggregation were made using the Cell Ranger software Version 5.0.0 Downstream analysis were performed on R studio using Seurat Supplementary files format and content: Tab separated values files and matrix files Assembly: Assembly: Lawson Annotation V4.3.2 Supplementary files format and content: 3 files of barcodes  features and matrix,FAC sorted cells,No treatments,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,Normal zebrafish growth condition,tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:Aktmutant,GSM8004755,GSM8004755: Zebrafish  EC  24hpf  Aktmutant; Danio rerio; RNA Seq,GSM8004755 r1,GSM8004755,1,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP482328,,loader:fastq load.py,mu1-23-19_DRT_S2_L001_I1_001.fastq.gz mu1-23-19_DRT_S2_L001_R1_001.fastq.gz mu1-23-19_DRT_S2_L001_R2_001.fastq.gz,fastq fastq fastq,23281472741.0,183318683.0,GSM8004755 r1,0:8 1:28 2:91,A:4703583066;C:3702175361;G:3998796644;T:4271932211;N:5512871,8,28,91,,4703583066,3702175361,3998796644,4271932211,5512871,SRX23122487,SRS20076037,SRA1780571,"Nicoli lab, School of Medicine, Yale University","Nicoli lab, School of Medicine, Yale University",,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-01-05,Pharyngula,Embryo,Multi-tissue,Multi-system
29809,SRR27450808,SRX23122487,SRS20076037,SRP482328,PRJNA1061565,Akt is the main kinase mediating the embryonic specification of artery cells,GSE252648,Transcriptome Analysis,This study aimed at understanding the function of Akt signaling in vascular development in Zebrafish. Overall design: This study used Zebrafish endothelial cells sorted from WT and akt full mutant embryos at 24hpf. Endothelial cells were sorted via the use of transgenic line kdrl:mCherry+. Only mCherry+ were sequenced. So here you can find sample WT and sample akt mutant.,,pubmed:39101673,,Zebrafish  EC  24hpf  Aktmutant,GSM8004755,,source name:FAC sorted cells|tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:Aktmutant|geo loc name:missing|collection date:missing,Zebrafish  EC  24hpf  Aktmutant,The barcoded processing  gene counting and aggregation were made using the Cell Ranger software Version 5.0.0 Downstream analysis were performed on R studio using Seurat Supplementary files format and content: Tab separated values files and matrix files Assembly: Assembly: Lawson Annotation V4.3.2 Supplementary files format and content: 3 files of barcodes  features and matrix,FAC sorted cells,No treatments,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,Normal zebrafish growth condition,tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:Aktmutant,GSM8004755,GSM8004755: Zebrafish  EC  24hpf  Aktmutant; Danio rerio; RNA Seq,GSM8004755 r1,GSM8004755,1,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP482328,,loader:fastq load.py,mu1-23-19_DRT_S2_L002_I1_001.fastq.gz mu1-23-19_DRT_S2_L002_R1_001.fastq.gz mu1-23-19_DRT_S2_L002_R2_001.fastq.gz,fastq fastq fastq,21009317138.0,165427694.0,GSM8004755 r2,0:8 1:28 2:91,A:4231955850;C:3363548064;G:3603913589;T:3849411287;N:5091364,8,28,91,,4231955850,3363548064,3603913589,3849411287,5091364,SRX23122487,SRS20076037,SRA1780571,"Nicoli lab, School of Medicine, Yale University","Nicoli lab, School of Medicine, Yale University",,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-01-05,Pharyngula,Embryo,Multi-tissue,Multi-system
29810,SRR27450809,SRX23122486,SRS20076036,SRP482328,PRJNA1061565,Akt is the main kinase mediating the embryonic specification of artery cells,GSE252648,Transcriptome Analysis,This study aimed at understanding the function of Akt signaling in vascular development in Zebrafish. Overall design: This study used Zebrafish endothelial cells sorted from WT and akt full mutant embryos at 24hpf. Endothelial cells were sorted via the use of transgenic line kdrl:mCherry+. Only mCherry+ were sequenced. So here you can find sample WT and sample akt mutant.,,pubmed:39101673,,Zebrafish  EC  24hpf  WT,GSM8004754,,source name:FAC sorted cells|tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:WT|geo loc name:missing|collection date:missing,Zebrafish  EC  24hpf  WT,The barcoded processing  gene counting and aggregation were made using the Cell Ranger software Version 5.0.0 Downstream analysis were performed on R studio using Seurat Supplementary files format and content: Tab separated values files and matrix files Assembly: Assembly: Lawson Annotation V4.3.2 Supplementary files format and content: 3 files of barcodes  features and matrix,FAC sorted cells,No treatments,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,Normal zebrafish growth condition,tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:WT,GSM8004754,GSM8004754: Zebrafish  EC  24hpf  WT; Danio rerio; RNA Seq,GSM8004754 r1,GSM8004754,1,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP482328,,loader:fastq load.py,wt1-23-19_DRT_S1_L001_I1_001.fastq.gz wt1-23-19_DRT_S1_L001_R1_001.fastq.gz wt1-23-19_DRT_S1_L001_R2_001.fastq.gz,fastq fastq fastq,22261273232.0,175285616.0,GSM8004754 r1,0:8 1:28 2:91,A:4475749188;C:3578353646;G:3836029871;T:4055646794;N:5211557,8,28,91,,4475749188,3578353646,3836029871,4055646794,5211557,SRX23122486,SRS20076036,SRA1780571,"Nicoli lab, School of Medicine, Yale University","Nicoli lab, School of Medicine, Yale University",,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-01-05,Pharyngula,Embryo,Multi-tissue,Multi-system
29811,SRR27450810,SRX23122486,SRS20076036,SRP482328,PRJNA1061565,Akt is the main kinase mediating the embryonic specification of artery cells,GSE252648,Transcriptome Analysis,This study aimed at understanding the function of Akt signaling in vascular development in Zebrafish. Overall design: This study used Zebrafish endothelial cells sorted from WT and akt full mutant embryos at 24hpf. Endothelial cells were sorted via the use of transgenic line kdrl:mCherry+. Only mCherry+ were sequenced. So here you can find sample WT and sample akt mutant.,,pubmed:39101673,,Zebrafish  EC  24hpf  WT,GSM8004754,,source name:FAC sorted cells|tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:WT|geo loc name:missing|collection date:missing,Zebrafish  EC  24hpf  WT,The barcoded processing  gene counting and aggregation were made using the Cell Ranger software Version 5.0.0 Downstream analysis were performed on R studio using Seurat Supplementary files format and content: Tab separated values files and matrix files Assembly: Assembly: Lawson Annotation V4.3.2 Supplementary files format and content: 3 files of barcodes  features and matrix,FAC sorted cells,No treatments,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell 3’ Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,Normal zebrafish growth condition,tissue:FAC sorted cells|cell line:kdrl:mCherry+|cell type:Endothelial Cells|genotype:WT,GSM8004754,GSM8004754: Zebrafish  EC  24hpf  WT; Danio rerio; RNA Seq,GSM8004754 r1,GSM8004754,1,Wild type  aktmutant  Tgkdrl:mCherry tissue  were dissected at 24 hpf. Dissected  tissues were dissociated into single cell suspensions and subjected to FACS.mCherry+ cells  which had 85% cell viability  were loaded onto the 10X Genomics Chromium instrument for a targeted recovery of 10 000 cells per sample. 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions 10X Genomics Chromium Next GEM Single Cell three prime Library Construction Kit V3.1 CG000204 was used to generate libraires according to manufacturer intructions Transcriptomic single cell  10x genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP482328,,loader:fastq load.py,wt1-23-19_DRT_S1_L002_I1_001.fastq.gz wt1-23-19_DRT_S1_L002_R1_001.fastq.gz wt1-23-19_DRT_S1_L002_R2_001.fastq.gz,fastq fastq fastq,20084395188.0,158144844.0,GSM8004754 r2,0:8 1:28 2:91,A:4028678970;C:3250176957;G:3455600305;T:3651884457;N:4840115,8,28,91,,4028678970,3250176957,3455600305,3651884457,4840115,SRX23122486,SRS20076036,SRA1780571,"Nicoli lab, School of Medicine, Yale University","Nicoli lab, School of Medicine, Yale University",,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-01-05,Pharyngula,Embryo,Multi-tissue,Multi-system
43987,SRR6811828,SRX3768868,SRS3023414,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 3 exo scar,GSM3032171,,source name:Pancreas except primary islet  liver|strain/background:Zebrabow M|tissue:Pancreas except primary islet  liver|developmental stage:Adult,Pancreas 3 exo scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Pancreas except primary islet  liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas except primary islet  liver|developmental stage:Adult,GSM3032171,GSM3032171: Pancreas 3 exo scar; Danio rerio; OTHER,GSM3032171,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM3032171,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P7exo_scar_R1.fastq.gz P7exo_scar_R2.fastq.gz,fastq fastq,4291410352.0,34608148.0,GSM3032171 r1,0:26 1:98,A:1266869162;C:1345773385;G:950601988;T:726078376;N:2087441,26,98,,,1266869162,1345773385,950601988,726078376,2087441,SRX3768868,SRS3023414,SRA623333,GEO,Max Delbrück Center,2,0.00014,0.00241,0.00012,0.00024,0.99995,0.99691,0.0,0.65306,26,98,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2018-03-06,Adult,Adult,Multi-tissue,Multi-system
43989,SRR6811826,SRX3768866,SRS3023382,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 3 scar,GSM3032169,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 3 scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM3032169,GSM3032169: Heart 3 scar; Danio rerio; OTHER,GSM3032169,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM3032169,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H7_scar_R1.fastq.gz H7_scar_R2.fastq.gz,fastq fastq,3999397296.0,32253204.0,GSM3032169 r1,0:26 1:98,A:1170970784;C:1255843391;G:881154893;T:689473188;N:1955040,26,98,,,1170970784,1255843391,881154893,689473188,1955040,SRX3768866,SRS3023382,SRA623333,GEO,Max Delbrück Center,2,0.00018,0.00228,0.00015,0.00019,0.99991,0.99659,0.8,0.67235,26,98,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2018-03-06,Adult,Adult,Multi-tissue,Multi-system
43993,SRR6811822,SRX3768862,SRS3023379,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 3 exo mRNA,GSM3032165,,source name:Pancreas except primary islet  liver|strain/background:Zebrabow M|tissue:Pancreas except primary islet  liver|developmental stage:Adult,Pancreas 3 exo mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 of publication. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Pancreas except primary islet  liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas except primary islet  liver|developmental stage:Adult,GSM3032165,GSM3032165: Pancreas 3 exo mRNA; Danio rerio; RNA Seq,GSM3032165,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM3032165,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P7exo_wt_R1.fastq.gz P7exo_wt_R2.fastq.gz,fastq fastq,38574026764.0,311080861.0,GSM3032165 r1,0:26 1:98,A:10751580091;C:8744299142;G:9264760851;T:9795905887;N:17480793,26,98,,,10751580091,8744299142,9264760851,9795905887,17480793,SRX3768862,SRS3023379,SRA623333,GEO,Max Delbrück Center,2,0.00463,0.93239,0.00336,0.04555,0.99671,0.87207,0.46341,0.57662,26,98,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2018-03-06,Adult,Adult,Multi-tissue,Multi-system
43995,SRR6811820,SRX3768860,SRS3023377,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 3 mRNA,GSM3032163,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 3 mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 of publication. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM3032163,GSM3032163: Heart 3 mRNA; Danio rerio; RNA Seq,GSM3032163,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM3032163,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H7_wt_R2.fastq.gz H7_wt_R1.fastq.gz,fastq fastq,45826897248.0,369571752.0,GSM3032163 r1,0:26 1:98,A:12856871948;C:10721924215;G:10335821801;T:11891517482;N:20761802,26,98,,,12856871948,10721924215,10335821801,11891517482,20761802,SRX3768860,SRS3023377,SRA623333,GEO,Max Delbrück Center,2,0.00303,0.90293,0.00206,0.05991,0.99746,0.86918,0.3421,0.66847,26,98,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2018-03-06,Adult,Adult,Multi-tissue,Multi-system
44000,SRR6211492,SRX3320767,SRS2626340,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 2 mRNA,GSM2830062,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 2 mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 in publication. Every sequencing read consists of a cellular barcode  a UMI  and a transcript sequence originating from an mRNA molecule. These transcript sequences were aligned using bwa aln3 with setting ' q 50' to a reference transcriptome constructed from Ensembl release 74 www.ensembl.org with extended three prime UTR regions. We filtered out all unmapped reads and all reads that were not uniquely mapped. post alignment  we determined which cellular barcodes corresponded to cells. We defined a cell to be a cellular barcode with at least five hundred uniquely mapped molecules. For each cellular barcode  we counted the number of molecules mapped to each gene  using the UMI correction method described by Grün et al. This method corrects for the possibility of the same UMI being used for two different transcripts in the same cell with the formula t =  K ln1 – k o/K  with t the final number of transcripts  k o the observed UMIs  and K the total number of UMIs possible. As protection against barcode sequencing errors  we counted the occurrence of each nucleotide for each barcode and filtered out barcodes in which one nucleotide occurred ten or more times. Furthermore  we filtered out barcodes that were one nucleotide substitution removed from a barcode with at least eight times as many transcripts. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM2830062,GSM2830062: Heart 2 mRNA; Danio rerio; RNA Seq,GSM2830062,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830062,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H6_wt_R2.fastq.gz H6_wt_R1.fastq.gz,fastq fastq,19466673438.0,141062851.0,GSM2830062 r1,0:28 1:110,A:5788453893;C:4266605110;G:4479895656;T:4930437501;N:1281278,28,110,,,5788453893,4266605110,4479895656,4930437501,1281278,SRX3320767,SRS2626340,SRA623333,GEO,Max Delbrück Center,2,0.00114,0.94531,0.00026,0.06835,0.99819,0.88308,0.68098,0.70993,28,110,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44001,SRR6211491,SRX3320766,SRS2626339,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 2 mRNA,GSM2830061,,source name:Pancreas and liver|strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,Pancreas 2 mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 in publication. Every sequencing read consists of a cellular barcode  a UMI  and a transcript sequence originating from an mRNA molecule. These transcript sequences were aligned using bwa aln3 with setting ' q 50' to a reference transcriptome constructed from Ensembl release 74 www.ensembl.org with extended three prime UTR regions. We filtered out all unmapped reads and all reads that were not uniquely mapped. post alignment  we determined which cellular barcodes corresponded to cells. We defined a cell to be a cellular barcode with at least five hundred uniquely mapped molecules. For each cellular barcode  we counted the number of molecules mapped to each gene  using the UMI correction method described by Grün et al. This method corrects for the possibility of the same UMI being used for two different transcripts in the same cell with the formula t =  K ln1 – k o/K  with t the final number of transcripts  k o the observed UMIs  and K the total number of UMIs possible. As protection against barcode sequencing errors  we counted the occurrence of each nucleotide for each barcode and filtered out barcodes in which one nucleotide occurred ten or more times. Furthermore  we filtered out barcodes that were one nucleotide substitution removed from a barcode with at least eight times as many transcripts. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Pancreas and liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,GSM2830061,GSM2830061: Pancreas 2 mRNA; Danio rerio; RNA Seq,GSM2830061,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830061,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P6_wt_R1.fastq.gz P6_wt_R2.fastq.gz,fastq fastq,17563278078.0,127270131.0,GSM2830061 r1,0:28 1:110,A:5146142876;C:3830859355;G:4368254000;T:4216873712;N:1148135,28,110,,,5146142876,3830859355,4368254000,4216873712,1148135,SRX3320766,SRS2626339,SRA623333,GEO,Max Delbrück Center,2,0.0023,0.93516,0.00062,0.07613,0.99667,0.8798,0.62666,0.6399,28,110,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44002,SRR6211490,SRX3320765,SRS2626338,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 1 mRNA,GSM2830060,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 1 mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 in publication. Every sequencing read consists of a cellular barcode  a UMI  and a transcript sequence originating from an mRNA molecule. These transcript sequences were aligned using bwa aln3 with setting ' q 50' to a reference transcriptome constructed from Ensembl release 74 www.ensembl.org with extended three prime UTR regions. We filtered out all unmapped reads and all reads that were not uniquely mapped. post alignment  we determined which cellular barcodes corresponded to cells. We defined a cell to be a cellular barcode with at least five hundred uniquely mapped molecules. For each cellular barcode  we counted the number of molecules mapped to each gene  using the UMI correction method described by Grün et al. This method corrects for the possibility of the same UMI being used for two different transcripts in the same cell with the formula t =  K ln1 – k o/K  with t the final number of transcripts  k o the observed UMIs  and K the total number of UMIs possible. As protection against barcode sequencing errors  we counted the occurrence of each nucleotide for each barcode and filtered out barcodes in which one nucleotide occurred ten or more times. Furthermore  we filtered out barcodes that were one nucleotide substitution removed from a barcode with at least eight times as many transcripts. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM2830060,GSM2830060: Heart 1 mRNA; Danio rerio; RNA Seq,GSM2830060,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830060,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H5_wt_R1.fastq.gz H5_wt_R2.fastq.gz,fastq fastq,11175332616.0,88693116.0,GSM2830060 r1,0:26 1:100,A:3373517159;C:2470603501;G:2531314673;T:2798372007;N:1525276,26,100,,,3373517159,2470603501,2531314673,2798372007,1525276,SRX3320765,SRS2626338,SRA623333,GEO,Max Delbrück Center,2,0.00264,0.95153,0.00046,0.05429,0.99669,0.88958,0.57635,0.67876,26,100,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44004,SRR6211488,SRX3320763,SRS2626336,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 1 mRNA,GSM2830058,,source name:Pancreas and liver|strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,Pancreas 1 mRNA,Alignment and transcript counting of libraries were done using Cell Ranger 2.0.2. Cell numbers to be extracted were set at a minimum of 6000 but were increased if there were substantially more cells with more than 500 unique transcripts. Exact numbers can be found in Supplementary Table 1 in publication. Every sequencing read consists of a cellular barcode  a UMI  and a transcript sequence originating from an mRNA molecule. These transcript sequences were aligned using bwa aln3 with setting ' q 50' to a reference transcriptome constructed from Ensembl release 74 www.ensembl.org with extended three prime UTR regions. We filtered out all unmapped reads and all reads that were not uniquely mapped. post alignment  we determined which cellular barcodes corresponded to cells. We defined a cell to be a cellular barcode with at least five hundred uniquely mapped molecules. For each cellular barcode  we counted the number of molecules mapped to each gene  using the UMI correction method described by Grün et al. This method corrects for the possibility of the same UMI being used for two different transcripts in the same cell with the formula t =  K ln1 – k o/K  with t the final number of transcripts  k o the observed UMIs  and K the total number of UMIs possible. As protection against barcode sequencing errors  we counted the occurrence of each nucleotide for each barcode and filtered out barcodes in which one nucleotide occurred ten or more times. Furthermore  we filtered out barcodes that were one nucleotide substitution removed from a barcode with at least eight times as many transcripts. Genome build: GRCz10   release 90 Supplementary files format and content: * matrix.mtx: Single cell transcript count table; * barcodes.tsv: List of cell barcodes.,Pancreas and liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,GSM2830058,GSM2830058: Pancreas 1 mRNA; Danio rerio; RNA Seq,GSM2830058,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830058,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P5_wt_R1.fastq.gz P5_wt_R2.fastq.gz,fastq fastq,18116725914.0,143783539.0,GSM2830058 r1,0:26 1:100,A:5348935548;C:3950936592;G:4404689151;T:4409667387;N:2497236,26,100,,,5348935548,3950936592,4404689151,4409667387,2497236,SRX3320763,SRS2626336,SRA623333,GEO,Max Delbrück Center,2,0.00319,0.93498,0.00092,0.08656,0.9947,0.86097,0.45652,0.53817,26,100,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44008,SRR6211484,SRX3320759,SRS2626332,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 2 scar,GSM2830055,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 2 scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. In the fourth filtering step  we determined the distribution of reads for the scars we had kept so far. Based on this distribution  we set a cut off and filtered out the scars that did not have at least this number of reads. Finally  for each cell type  we determined the distribution of different scars seen per cell and set a maximum number of scars a cell of that type can have. We filtered out cells in which we observed more than this maximum number as possible doublets. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM2830055,GSM2830055: Heart 2 scar; Danio rerio; OTHER,GSM2830055,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830055,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H6_scar_R1.fastq.gz H6_scar_R2.fastq.gz,fastq fastq,2079107172.0,15065994.0,GSM2830055 r1,0:28 1:110,A:580640871;C:641740953;G:501505035;T:355092832;N:127481,28,110,,,580640871,641740953,501505035,355092832,127481,SRX3320759,SRS2626332,SRA623333,GEO,Max Delbrück Center,2,5e-05,0.00253,4e-05,0.00023,1.0,0.99778,,0.58419,28,110,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44009,SRR6211483,SRX3320758,SRS2626348,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 2 scar,GSM2830054,,source name:Pancreas and liver|strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,Pancreas 2 scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. In the fourth filtering step  we determined the distribution of reads for the scars we had kept so far. Based on this distribution  we set a cut off and filtered out the scars that did not have at least this number of reads. Finally  for each cell type  we determined the distribution of different scars seen per cell and set a maximum number of scars a cell of that type can have. We filtered out cells in which we observed more than this maximum number as possible doublets. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Pancreas and liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,GSM2830054,GSM2830054: Pancreas 2 scar; Danio rerio; OTHER,GSM2830054,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830054,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P6_scar_R1.fastq.gz P6_scar_R2.fastq.gz,fastq fastq,1026715860.0,7439970.0,GSM2830054 r1,0:28 1:110,A:287816843;C:320879355;G:243598630;T:174359452;N:61580,28,110,,,287816843,320879355,243598630,174359452,61580,SRX3320758,SRS2626348,SRA623333,GEO,Max Delbrück Center,2,0.00029,0.00123,0.00028,6e-05,1.0,0.99914,,0.25714,28,110,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44010,SRR6211482,SRX3320757,SRS2626331,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Heart 1 scar,GSM2830053,,source name:Heart and blood|strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,Heart 1 scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. In the fourth filtering step  we determined the distribution of reads for the scars we had kept so far. Based on this distribution  we set a cut off and filtered out the scars that did not have at least this number of reads. Finally  for each cell type  we determined the distribution of different scars seen per cell and set a maximum number of scars a cell of that type can have. We filtered out cells in which we observed more than this maximum number as possible doublets. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Heart and blood,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Heart and blood|developmental stage:Adult,GSM2830053,GSM2830053: Heart 1 scar; Danio rerio; OTHER,GSM2830053,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830053,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,H5_scar_R1.fastq.gz H5_scar_R2.fastq.gz,fastq fastq,1287546624.0,10218624.0,GSM2830053 r1,0:26 1:100,A:365272675;C:413037677;G:296495693;T:212570153;N:170426,26,100,,,365272675,413037677,296495693,212570153,170426,SRX3320757,SRS2626331,SRA623333,GEO,Max Delbrück Center,2,0.00023,0.00044,0.00022,3e-05,1.0,0.99955,,0.32653,26,100,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
44012,SRR6211480,SRX3320755,SRS2626329,SRP121343,PRJNA415636,Simultaneous lineage tracing and cell type identification using CRISPR/Cas9 induced genetic scars,GSE106121,Other,A key goal of developmental biology is to understand how a single cell transforms into a full grown organism consisting of many different cell types. Single cell RNA sequencing scRNA seq has become a widely used method due to its ability to identify all cell types in a tissue or organ in a systematic manner. However  a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here  we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes  we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses  LINNAEUS LINeage tracing by Nuclease Activated Editing of Ubiquitous Sequences can be used as a systematic approach for identifying the lineage origin of novel cell types  or of known cell types under different conditions. Overall design: Combining scRNA seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes.,,pubmed:29644996,,Pancreas 1 scar,GSM2830051,,source name:Pancreas and liver|strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,Pancreas 1 scar,Library strategy: Targeted amplification Scar reads have the same structure as transcript reads: they consist of a barcode  a UMI and a scar. The scar sequences were aligned using bwa mem6 to a reference of RFP. We defined a cell as a barcode with at least 500 reads. We removed reads that were unmapped  had an incorrect barcode  or did not start with the exact PCR primer we used. We truncated all scar sequences to 75 nucleotides and filtered out shorter sequences. To correct for sequencing errors  we implemented several rounds of scar filtering Supplementary Fig. 2 in publication. We started by counting the number of times each molecule was sequenced. Sequencing errors will typically have fewer reads than the actual scars they originate from. As a first filtering step  we therefore removed all molecules only seen once to reduce the complexity in the dataset for consecutive filtering steps. In the second filtering step  we aimed to remove easily recognizable sequencing errors. To this end  we consecutively considered scar sequences that have the same cellular barcode and UMI  UMIs that have the same cellular barcode and scar sequence  and cellular barcodes that have the same UMI and scar sequence. In each step  we kept only the molecule with the highest number of reads. The rationale behind this is that it is very improbable to have two valid scar sequences in the same cell with the same UMI  or to have a scar sequence with the same UMI appear in two different cells. The observation of two different UMIs for the same scar in the same cell is much more likely and corresponds to detection of multiple transcripts from the same locus  but information about scar expression levels was not required in our downstream analysis. In the third filtering step  we specifically targeted sequencing errors within each cell. We compared the scar sequences found within a cell to each other. We filtered out sequences that had a Hamming distance of 2 or less to another scar sequence in the same cell that occurred in at least eight times as many reads. Scar sequences in the same cell that were one Hamming distance apart but had a read ratio less than eight were tested on three criteria if both of them occurred at least twice in the scar library: Do both scars have more than one transcript?  Do both scars occur in cells independently from each other?  Do the UMIs of both scars have Hamming distance of two or more? If two of these criteria were true  the scars were kept and the sequences were placed on a list of validated scars that  if they occurred in the same cell in another library  did not have to be tested anymore. If one or zero criteria were true  the scar that had only one transcript  or the scar that did not occur independently  were filtered out. In the fourth filtering step  we determined the distribution of reads for the scars we had kept so far. Based on this distribution  we set a cut off and filtered out the scars that did not have at least this number of reads. Finally  for each cell type  we determined the distribution of different scars seen per cell and set a maximum number of scars a cell of that type can have. We filtered out cells in which we observed more than this maximum number as possible doublets. Genome build: N/A Supplementary files format and content: List of scar transcripts with cell barcode  scar name  cell type  scar probability and number of organisms that have this cell.,Pancreas and liver,,Single cell dissociation. 10X Genomics Chromium,,strain/background:Zebrabow M|tissue:Pancreas and liver|developmental stage:Adult,GSM2830051,GSM2830051: Pancreas 1 scar; Danio rerio; OTHER,GSM2830051,,1,Single cell dissociation. 10X Genomics Chromium,GEO Accession:GSM2830051,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP121343,,,P5_scar_R1.fastq.gz P5_scar_R2.fastq.gz,fastq fastq,1192602600.0,9465100.0,GSM2830051 r1,0:26 1:100,A:346841760;C:383266222;G:268916933;T:193424756;N:152929,26,100,,,346841760,383266222,268916933,193424756,152929,SRX3320755,SRS2626329,SRA623333,GEO,Max Delbrück Center,2,0.00021,0.00091,0.00014,2e-05,0.99997,0.99892,0.0,0.23931,26,100,T,T,mates < 9% mapping rate,illumina,nextseq,unknown,other,unknown,sc,single_cell_droplet,10x,,Germany,2017-10-24,Adult,Adult,Multi-tissue,Multi-system
53493,SRR9881612,SRX6634133,SRS5205093,SRP217216,PRJNA558070,N Glycome regulation limits the transdifferentiation of endothelial cells into hematopoietic stem cells,GSE135246,Transcriptome Analysis,Hematopoietic stem and progenitor cells HSPCs are required to establish and maintain the adult blood system in vertebrates. During development  HPSCs are generated from hemogenic endothelial cells that undergo an endothelial to hematopoietic transition EHT. Growth factors and epigenetic changes can promote EHT  but these mechanisms do not explain its tight spatiotemporal regulation during development. Here  we show that microRNA miR miR 223 mediated regulation of N glycan biosynthesis intrinsically restrains EHT  representing a new pathway that prevents excessive HSPC production. We find that miR 223 is uniquely expressed in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR 223 promotes the expansion of these cells in the zebrafish and mouse aorta gonad mesonephros AGM  where EHT occurs. miR 223 targets alg2 alpha 1 3/ 1 6 mannosyltransferase in the AGM endothelium to restrict hemogenic and HSPC specification. This enzyme is involved in the attachment of the N glycans   a common co translational modification9 12 that influences several pathophysiological processes  but has not yet been implicated in EHT. Using an N glycosensor  we demonstrate that abundant protein N glycan attachment occurs in vascular cells during EHT  and this process is required for HSPC production. High throughput glycome analysis upon loss of miR 223 revealed that terminal alpha 1 3/6 mannose modifications are increased at the expense of alpha 2 3/6 sialic acid sugars. Importantly  pharmacological manipulation targeting these N glycan types in wild type embryos phenocopies the loss of miR 223 and enhances EHT as well as HSPC production. Thus  the N glycome plays a previously unappreciated role as an intrinsic regulator of EHT  with specific mannose and sialic acid modifications serving as key endothelial determinants to restrict the hematopoietic fate. Overall design: Single cell RNA sequencing for zebrafish Tgkdrl:hras mCherrys896 endothelial cells at 27 hpf for wildtype and miR 223ya303 backgrounds,,pubmed:33273096,,223mut 27hpf EC1,GSM3996907,,source name:endothelial cell|tissue:trunk endothelium|cell type:endothelial cell|developmental stage:27 hpf 223:KalTA4ya363; Tg5xUAS:eGFPnkuasgfp1a; Tgkdrl:hras mCherrys896; miR 223ya303|genotype:223mut,223mut 27hpf EC1,The reads were demultiplexed using bcl2fastq provided by the 10X genomics.  The demultiplexed reads were aligned to the reference genome Grz11  modified to add mCherry  GAL4  and eGFP to obtain digital gene expression matrix data using cellranger program The output of the cellranger was then processed using Seurat in R. All the cells with less than 200 unique transcripts were eliminared.  All genes which are expressed in less than 3 cells were also eliminated. The data was normalized using sctransform  and then clustered.  The clusters were projected for viualization in R using the PHATE algorithm. Genome build: GRCz11 Supplementary files format and content: Average Gene expression matrix for average gene expression per cluster  per condition.  This is a tab separated values file  where each row represents a gene  and each column represents the cluster  and the condition  and each cell represents normalized expression values across all the cells in that cluster for that condition.,endothelial cell,,Trunk tissue was dissected from 27 hpf zebrafish embryos under  0.1% tricaine anesthesia and then disassociated into a single cell suspension using 0.25 mg/ml liberase for 45 minutes. Tgkdrl:hras mCherrys896 endothelial cells were isolated from the single cell suspension via FACS into 0.04% BSA in PBS and applied to the 10X genomics single cell RNA sequencing platform. 10 X genomics Chromium Next GEM Single Cell 3’ Library Construction Kit v2.1,Zebrafish were raised and maintained at 28.5˚C using standard methods and according to protocols approved by Yale University Institutional Animal Care and Use Committee # 2017 11473.,tissue:trunk endothelium|cell type:endothelial cell|developmental stage:27 hpf 223:KalTA4ya363; Tg5xUAS:eGFPnkuasgfp1a; Tgkdrl:hras mCherrys896; miR 223ya303|genotype:223mut,GSM3996907,GSM3996907: 223mut 27hpf EC1; Danio rerio; RNA Seq,GSM3996907,,1,Trunk tissue was dissected from 27 hpf zebrafish embryos under  0.1% tricaine anesthesia and then disassociated into a single cell suspension using 0.25 mg/ml liberase for 45 minutes. Tgkdrl:hras mCherrys896 endothelial cells were isolated from the single cell suspension via FACS into 0.04% BSA in PBS and applied to the 10X genomics single cell RNA sequencing platform. 10 X genomics Chromium Next GEM Single Cell three prime Library Construction Kit v2.1,GEO Accession:GSM3996907,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP217216,,,Mut_pssorted_genome_bam.bam,10X Genomics bam file,31174899854.0,318111223.0,GSM3996907 r1,0:98,A:8464403532;C:7291172763;G:7282393533;T:8131647604;N:5282422,98,,,,8464403532,7291172763,7282393533,8131647604,5282422,SRX6634133,SRS5205093,SRA930499,GEO,"Internal Medicine, Yale University",1,0.92745,,0.05306,,0.84502,,0.46383,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,small_rna,unknown,sc,single_cell_droplet,10x,,United States,2019-08-01,Pharyngula,Embryo,Multi-tissue,Multi-system
53494,SRR9881611,SRX6634132,SRS5205092,SRP217216,PRJNA558070,N Glycome regulation limits the transdifferentiation of endothelial cells into hematopoietic stem cells,GSE135246,Transcriptome Analysis,Hematopoietic stem and progenitor cells HSPCs are required to establish and maintain the adult blood system in vertebrates. During development  HPSCs are generated from hemogenic endothelial cells that undergo an endothelial to hematopoietic transition EHT. Growth factors and epigenetic changes can promote EHT  but these mechanisms do not explain its tight spatiotemporal regulation during development. Here  we show that microRNA miR miR 223 mediated regulation of N glycan biosynthesis intrinsically restrains EHT  representing a new pathway that prevents excessive HSPC production. We find that miR 223 is uniquely expressed in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR 223 promotes the expansion of these cells in the zebrafish and mouse aorta gonad mesonephros AGM  where EHT occurs. miR 223 targets alg2 alpha 1 3/ 1 6 mannosyltransferase in the AGM endothelium to restrict hemogenic and HSPC specification. This enzyme is involved in the attachment of the N glycans   a common co translational modification9 12 that influences several pathophysiological processes  but has not yet been implicated in EHT. Using an N glycosensor  we demonstrate that abundant protein N glycan attachment occurs in vascular cells during EHT  and this process is required for HSPC production. High throughput glycome analysis upon loss of miR 223 revealed that terminal alpha 1 3/6 mannose modifications are increased at the expense of alpha 2 3/6 sialic acid sugars. Importantly  pharmacological manipulation targeting these N glycan types in wild type embryos phenocopies the loss of miR 223 and enhances EHT as well as HSPC production. Thus  the N glycome plays a previously unappreciated role as an intrinsic regulator of EHT  with specific mannose and sialic acid modifications serving as key endothelial determinants to restrict the hematopoietic fate. Overall design: Single cell RNA sequencing for zebrafish Tgkdrl:hras mCherrys896 endothelial cells at 27 hpf for wildtype and miR 223ya303 backgrounds,,pubmed:33273096,,wildtype 27hpf EC1,GSM3996906,,source name:endothelial cell|tissue:trunk endothelium|cell type:endothelial cell|developmental stage:27 hpf 223:KalTA4ya362; Tg5xUAS:eGFPnkuasgfp1a; Tgkdrl:hras mCherrys896|genotype:wildtype,wildtype 27hpf EC1,The reads were demultiplexed using bcl2fastq provided by the 10X genomics.  The demultiplexed reads were aligned to the reference genome Grz11  modified to add mCherry  GAL4  and eGFP to obtain digital gene expression matrix data using cellranger program The output of the cellranger was then processed using Seurat in R. All the cells with less than 200 unique transcripts were eliminared.  All genes which are expressed in less than 3 cells were also eliminated. The data was normalized using sctransform  and then clustered.  The clusters were projected for viualization in R using the PHATE algorithm. Genome build: GRCz11 Supplementary files format and content: Average Gene expression matrix for average gene expression per cluster  per condition.  This is a tab separated values file  where each row represents a gene  and each column represents the cluster  and the condition  and each cell represents normalized expression values across all the cells in that cluster for that condition.,endothelial cell,,Trunk tissue was dissected from 27 hpf zebrafish embryos under  0.1% tricaine anesthesia and then disassociated into a single cell suspension using 0.25 mg/ml liberase for 45 minutes. Tgkdrl:hras mCherrys896 endothelial cells were isolated from the single cell suspension via FACS into 0.04% BSA in PBS and applied to the 10X genomics single cell RNA sequencing platform. 10 X genomics Chromium Next GEM Single Cell 3’ Library Construction Kit v2.1,Zebrafish were raised and maintained at 28.5˚C using standard methods and according to protocols approved by Yale University Institutional Animal Care and Use Committee # 2017 11473.,tissue:trunk endothelium|cell type:endothelial cell|developmental stage:27 hpf 223:KalTA4ya362; Tg5xUAS:eGFPnkuasgfp1a; Tgkdrl:hras mCherrys896|genotype:wildtype,GSM3996906,GSM3996906: wildtype 27hpf EC1; Danio rerio; RNA Seq,GSM3996906,,1,Trunk tissue was dissected from 27 hpf zebrafish embryos under  0.1% tricaine anesthesia and then disassociated into a single cell suspension using 0.25 mg/ml liberase for 45 minutes. Tgkdrl:hras mCherrys896 endothelial cells were isolated from the single cell suspension via FACS into 0.04% BSA in PBS and applied to the 10X genomics single cell RNA sequencing platform. 10 X genomics Chromium Next GEM Single Cell three prime Library Construction Kit v2.1,GEO Accession:GSM3996906,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 4000,,SRP217216,,,WT_pssorted_genome_bam.bam,10X Genomics bam file,26866951370.0,274152565.0,GSM3996906 r1,0:98,A:7373453026;C:6169004304;G:6196779570;T:7121490110;N:6224360,98,,,,7373453026,6169004304,6196779570,7121490110,6224360,SRX6634132,SRS5205092,SRA930499,GEO,"Internal Medicine, Yale University",1,0.93015,,0.0602,,0.83853,,0.4683,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,small_rna,unknown,sc,single_cell_droplet,10x,,United States,2019-08-01,Pharyngula,Embryo,Multi-tissue,Multi-system
56706,SRR11040640,SRX7692417,SRS6118510,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3lof/lof,GSM4301251,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,2 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301251,GSM4301251: 2 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301251,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301251,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3lof-R3-ZF-0519_S3_L001_R1_001.fastq.gz 2-fgfr3lof-R3-ZF-0519_S3_L001_R2_001.fastq.gz,fastq fastq,2956636039.0,24845681.0,GSM4301251 r1,0:28 1:91,A:805752236;C:683372886;G:699894274;T:767405768;N:210875,28,91,,,805752236,683372886,699894274,767405768,210875,SRX7692417,SRS6118510,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00944,0.95293,0.0032,0.11845,0.98863,0.79573,0.31275,0.48226,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56707,SRR11040641,SRX7692417,SRS6118510,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3lof/lof,GSM4301251,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,2 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301251,GSM4301251: 2 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301251,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301251,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3lof-R3-ZF-0519_S4_L001_R1_001.fastq.gz 2-fgfr3lof-R3-ZF-0519_S4_L001_R2_001.fastq.gz,fastq fastq,2943149888.0,24732352.0,GSM4301251 r2,0:28 1:91,A:801299410;C:680335967;G:696876648;T:764428085;N:209778,28,91,,,801299410,680335967,696876648,764428085,209778,SRX7692417,SRS6118510,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00982,0.95341,0.00295,0.11853,0.98798,0.79429,0.32383,0.48491,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56708,SRR11040642,SRX7692417,SRS6118510,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3lof/lof,GSM4301251,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,2 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301251,GSM4301251: 2 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301251,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301251,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3lof-R3-ZF-0519_S8_L001_R1_001.fastq.gz 2-fgfr3lof-R3-ZF-0519_S8_L001_R2_001.fastq.gz,fastq fastq,2930322283.0,24624557.0,GSM4301251 r3,0:28 1:91,A:800017449;C:676370110;G:692286275;T:761438555;N:209894,28,91,,,800017449,676370110,692286275,761438555,209894,SRX7692417,SRS6118510,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.0091,0.95344,0.00277,0.11835,0.98863,0.79523,0.30888,0.47399,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56709,SRR11040643,SRX7692417,SRS6118510,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3lof/lof,GSM4301251,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,2 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301251,GSM4301251: 2 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301251,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301251,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3lof-R3-ZF-0519_S10_L001_R1_001.fastq.gz 2-fgfr3lof-R3-ZF-0519_S10_L001_R2_001.fastq.gz,fastq fastq,3086393758.0,25936082.0,GSM4301251 r4,0:28 1:91,A:842498816;C:712567363;G:729291108;T:801817022;N:219449,28,91,,,842498816,712567363,729291108,801817022,219449,SRX7692417,SRS6118510,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00907,0.95227,0.00303,0.11898,0.98906,0.79555,0.32194,0.48998,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56710,SRR11040636,SRX7692416,SRS6118508,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3lof/lof,GSM4301250,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,1 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301250,GSM4301250: 1 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301250,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301250,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3lof-R3-ZF-0519_S2_L001_R1_001.fastq.gz 1-fgfr3lof-R3-ZF-0519_S2_L001_R2_001.fastq.gz,fastq fastq,3809919113.0,32016127.0,GSM4301250 r1,0:28 1:91,A:1051323356;C:870053361;G:886403932;T:1001865374;N:273090,28,91,,,1051323356,870053361,886403932,1001865374,273090,SRX7692416,SRS6118508,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.0073,0.95159,0.00239,0.13839,0.98979,0.78293,0.32315,0.49047,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56711,SRR11040637,SRX7692416,SRS6118508,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3lof/lof,GSM4301250,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,1 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301250,GSM4301250: 1 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301250,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301250,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3lof-R3-ZF-0519_S5_L001_R1_001.fastq.gz 1-fgfr3lof-R3-ZF-0519_S5_L001_R2_001.fastq.gz,fastq fastq,5231239643.0,43959997.0,GSM4301250 r2,0:28 1:91,A:1444902743;C:1193884261;G:1216010955;T:1376068197;N:373487,28,91,,,1444902743,1193884261,1216010955,1376068197,373487,SRX7692416,SRS6118508,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00703,0.95125,0.00257,0.13933,0.99062,0.78417,0.33564,0.4777,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56712,SRR11040638,SRX7692416,SRS6118508,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3lof/lof,GSM4301250,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,1 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301250,GSM4301250: 1 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301250,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301250,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3lof-R3-ZF-0519_S14_L001_R1_001.fastq.gz 1-fgfr3lof-R3-ZF-0519_S14_L001_R2_001.fastq.gz,fastq fastq,6034697060.0,50711740.0,GSM4301250 r3,0:28 1:91,A:1667295741;C:1376061258;G:1401956669;T:1588951873;N:431519,28,91,,,1667295741,1376061258,1401956669,1588951873,431519,SRX7692416,SRS6118508,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00707,0.94822,0.00239,0.13943,0.99038,0.78311,0.36655,0.47115,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56713,SRR11040639,SRX7692416,SRS6118508,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3lof/lof,GSM4301250,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,1 fgfr3lof/lof,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3lof1/lof1|length:9 mm,GSM4301250,GSM4301250: 1 fgfr3lof/lof; Danio rerio; RNA Seq,GSM4301250,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301250,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3lof-R3-ZF-0519_S15_L001_R1_001.fastq.gz 1-fgfr3lof-R3-ZF-0519_S15_L001_R2_001.fastq.gz,fastq fastq,5425038164.0,45588556.0,GSM4301250 r4,0:28 1:91,A:1498130539;C:1238395410;G:1261876829;T:1426249730;N:385656,28,91,,,1498130539,1238395410,1261876829,1426249730,385656,SRX7692416,SRS6118508,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00733,0.94973,0.0026,0.13793,0.99068,0.78423,0.3337,0.48233,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56714,SRR11040632,SRX7692415,SRS6118509,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3+/+,GSM4301249,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,2 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301249,GSM4301249: 2 fgfr3+/+; Danio rerio; RNA Seq,GSM4301249,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301249,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3plus-R3-ZF-0519_S6_L001_R1_001.fastq.gz 2-fgfr3plus-R3-ZF-0519_S6_L001_R2_001.fastq.gz,fastq fastq,4962416501.0,41700979.0,GSM4301249 r1,0:28 1:91,A:1356709063;C:1139750321;G:1170875983;T:1294727572;N:353562,28,91,,,1356709063,1139750321,1170875983,1294727572,353562,SRX7692415,SRS6118509,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00727,0.95235,0.00248,0.13046,0.98981,0.78054,0.33659,0.48621,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56715,SRR11040633,SRX7692415,SRS6118509,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3+/+,GSM4301249,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,2 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301249,GSM4301249: 2 fgfr3+/+; Danio rerio; RNA Seq,GSM4301249,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301249,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3plus-R3-ZF-0519_S12_L001_R1_001.fastq.gz 2-fgfr3plus-R3-ZF-0519_S12_L001_R2_001.fastq.gz,fastq fastq,6695697316.0,56266364.0,GSM4301249 r2,0:28 1:91,A:1829441811;C:1538021924;G:1580312219;T:1747444971;N:476391,28,91,,,1829441811,1538021924,1580312219,1747444971,476391,SRX7692415,SRS6118509,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00744,0.95393,0.0026,0.13086,0.98969,0.78139,0.34081,0.50162,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56716,SRR11040634,SRX7692415,SRS6118509,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3+/+,GSM4301249,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,2 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301249,GSM4301249: 2 fgfr3+/+; Danio rerio; RNA Seq,GSM4301249,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301249,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3plus-R3-ZF-0519_S13_L001_R1_001.fastq.gz 2-fgfr3plus-R3-ZF-0519_S13_L001_R2_001.fastq.gz,fastq fastq,4853594809.0,40786511.0,GSM4301249 r3,0:28 1:91,A:1328029955;C:1113808349;G:1144735618;T:1266673333;N:347554,28,91,,,1328029955,1113808349,1144735618,1266673333,347554,SRX7692415,SRS6118509,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00737,0.95245,0.00274,0.12891,0.99038,0.78228,0.3487,0.4977,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56717,SRR11040635,SRX7692415,SRS6118509,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,2 fgfr3+/+,GSM4301249,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,2 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301249,GSM4301249: 2 fgfr3+/+; Danio rerio; RNA Seq,GSM4301249,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301249,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,2-fgfr3plus-R3-ZF-0519_S16_L001_R1_001.fastq.gz 2-fgfr3plus-R3-ZF-0519_S16_L001_R2_001.fastq.gz,fastq fastq,6323597406.0,53139474.0,GSM4301249 r4,0:28 1:91,A:1727515649;C:1453521438;G:1493844843;T:1648265752;N:449724,28,91,,,1727515649,1453521438,1493844843,1648265752,449724,SRX7692415,SRS6118509,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00737,0.95341,0.00266,0.12818,0.98934,0.78151,0.32417,0.4918,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56718,SRR11040628,SRX7692414,SRS6118507,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3+/+,GSM4301248,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,1 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301248,GSM4301248: 1 fgfr3+/+; Danio rerio; RNA Seq,GSM4301248,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301248,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3plus-R3-ZF-0519_S1_L001_R1_001.fastq.gz 1-fgfr3plus-R3-ZF-0519_S1_L001_R2_001.fastq.gz,fastq fastq,6286029939.0,52823781.0,GSM4301248 r1,0:28 1:91,A:1726222024;C:1448493933;G:1483478967;T:1627385939;N:449076,28,91,,,1726222024,1448493933,1483478967,1627385939,449076,SRX7692414,SRS6118507,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00651,0.95215,0.0021,0.11197,0.99099,0.79914,0.32593,0.49237,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56719,SRR11040629,SRX7692414,SRS6118507,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3+/+,GSM4301248,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,1 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301248,GSM4301248: 1 fgfr3+/+; Danio rerio; RNA Seq,GSM4301248,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301248,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3plus-R3-ZF-0519_S7_L001_R1_001.fastq.gz 1-fgfr3plus-R3-ZF-0519_S7_L001_R2_001.fastq.gz,fastq fastq,5384386336.0,45246944.0,GSM4301248 r2,0:28 1:91,A:1482057664;C:1241359922;G:1266945881;T:1393638700;N:384169,28,91,,,1482057664,1241359922,1266945881,1393638700,384169,SRX7692414,SRS6118507,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00644,0.9527,0.00205,0.11156,0.99111,0.80217,0.3568,0.48853,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56720,SRR11040630,SRX7692414,SRS6118507,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3+/+,GSM4301248,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,1 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301248,GSM4301248: 1 fgfr3+/+; Danio rerio; RNA Seq,GSM4301248,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301248,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3plus-R3-ZF-0519_S9_L001_R1_001.fastq.gz 1-fgfr3plus-R3-ZF-0519_S9_L001_R2_001.fastq.gz,fastq fastq,5916050133.0,49714707.0,GSM4301248 r3,0:28 1:91,A:1628484212;C:1362413135;G:1391038139;T:1533691145;N:423502,28,91,,,1628484212,1362413135,1391038139,1533691145,423502,SRX7692414,SRS6118507,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.0066,0.95312,0.00209,0.11367,0.99109,0.79857,0.336,0.47399,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
56721,SRR11040631,SRX7692414,SRS6118507,SRP247644,PRJNA605419,FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development,GSE144944,Transcriptome Analysis,In order to study FGFRthree primes role in cranial vault development  we generated the first fgfr3 loss of function zebrafish fgfr3lof1/lof1. The mutant fish exhibited major changes in the craniofacial skeleton  with a lack of sutures  abnormal frontal and parietal bones  and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype  we performed single cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell cell communication  mineralization  and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377  we profiled the transcriptome of nearly 28000 single cells using the Chromium system 10x Genomics. Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types osteogenic cells  chondrogenic cells  immune cells epidermal cells  endothelial cells. The scRNA seq libraries were generated with a Chromium Single Cell three prime Library & Gel Bead Kit v.3 10x Genomics. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,pubmed:32379366,,1 fgfr3+/+,GSM4301248,,source name:Cranial vault|tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,1 fgfr3+/+,Fastq files from the scRNA 10X libraries were processed using the Cell Ranger Count pipeline with its default parameters. Reads were aligned against the GRCz11 3.0.0 reference transcriptome. The RNA data quality control and downstream analysis were performed using the Seurat R package version 3.0.2 and the standard Seurat v3 integration workflow. We filtered cells that had i unique feature counts over 2000 or below 500  or ii more than 20% of mitochondrial counts. post data filtering  we obtained an expression matrix with 21176 genes and 19245 cells. The matrix’s dimensions were reduced by running the 20 significant principal components against scaled data. Corresponding to an object of class Seurat SC fgfr3 Cranial Vault ZF combined samples. We used the FindMarkers function to define marker of each cluster Findmarkers by cluster.xlsx file. We used the FindMarkers function to compute differentially expressed genes DEGs for each of the identity classes in the dataset DEGS.xlsx file. We subset cluster 4 from SC fgfr3 Cranial Vault ZF combined samples matrix and performed new clustering resolution 0.2 and we obtained SC fgfr3 Cranial Vault ZF Subset Cluster4. Genome build: GRCz11 3.0.0 Supplementary files format and content: * raw feature bc matrix.h5 Supplementary files format and content: Findmarkers by cluster.xlsx Supplementary files format and content: DEGS.xlsx,Cranial vault,,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer’s instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,,tissue:Cranial vault|genotype/variation:fgfr3+/+|length:9 mm,GSM4301248,GSM4301248: 1 fgfr3+/+; Danio rerio; RNA Seq,GSM4301248,,1,The cranial vault was isolated from the head of 9SL zebrafish and dissected in PBS. The olfactory bulbs  the anterior parts of the frontal bones  and the suboccipital bones were removed. The soft tissue was partially degraded by incubation with 0.2% collagenase and 4 mM EDTA in PBS for 10 min at 28°C. post a wash in PBS  cells were dissociated for 40 min at 28°C in 0.2% collagenase in PBS. The solution was strained using a 40 μm cell strainer  and the cells were washed in a MEM supplement with 10% FBS. Prior to flow cytometry  the cells were suspended in FacsMax and stained with SytoxTM green nucleic acid stain and Hoechst. To sort the cells  beads were used to select events larger than 3 µm. Hoechst positive cells cells with a nucleus and Syber green negative cells live cells were sorted. The scRNA seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 10x Genomics  according to the manufacturer's instructions. Briefly  cells were counted  diluted at 1000 cells/µL in PBS+0.04% BSA  and 6500 cells were loaded in the 10x Chromium Controller to generate single cell gel beads in an emulsion. post reverse transcription  the gel beads in the emulsion were disrupted. Barcoded complementary DNA was isolated and amplified in PCRs. Following fragmentation  end repair and A tailing  sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq 6000 Illumina on paired end strands read 1: 28bp; read 2: 91bp  with a mean read depth of 25000 reads per cell.,GEO Accession:GSM4301248,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP247644,,,1-fgfr3plus-R3-ZF-0519_S11_L001_R1_001.fastq.gz 1-fgfr3plus-R3-ZF-0519_S11_L001_R2_001.fastq.gz,fastq fastq,5591429319.0,46986801.0,GSM4301248 r4,0:28 1:91,A:1539075154;C:1286758672;G:1316467634;T:1448729317;N:398542,28,91,,,1539075154,1286758672,1316467634,1448729317,398542,SRX7692414,SRS6118507,SRA1039424,GEO,"Molecular and physiopathological bases of osteochondrodysplasia, U1163, Institut Imagine",2,0.00664,0.95136,0.00216,0.11396,0.99131,0.8003,0.32678,0.49629,28,91,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,sc,single_cell_droplet,10x,,France,2020-02-07,Undetermined,Undetermined,Multi-tissue,Multi-system
59196,SRR11777188,SRX8330063,SRS6649550,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 3,GSM4547702,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547702,GSM4547702: dominant 3; Danio rerio; RNA Seq,GSM4547702,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547702,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample8 I1.fastq.gz   read2PairFiles=Next sample8 R1.fastq.gz   read3PairFiles=Next sample8 R2.fastq.gz,Next_sample8_I1.fastq.gz Next_sample8_R1.fastq.gz Next_sample8_R2.fastq.gz,fastq fastq fastq,6949629860.0,75539455.0,GSM4547702 r1,0:8 1:26 2:58,A:1844979631;C:1603125876;G:1713257721;T:1782703901;N:5562731,8,26,58,,1844979631,1603125876,1713257721,1782703901,5562731,SRX8330063,SRS6649550,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95614,,0.07196,,0.85527,,0.53876,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59197,SRR11777189,SRX8330063,SRS6649550,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 3,GSM4547702,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547702,GSM4547702: dominant 3; Danio rerio; RNA Seq,GSM4547702,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547702,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample8 S8 L001 I1 001.fastq.gz   read2PairFiles=sample8 S8 L001 R1 001.fastq.gz   read3PairFiles=sample8 S8 L001 R2 001.fastq.gz,sample8_S8_L001_R2_001.fastq.gz sample8_S8_L001_R1_001.fastq.gz sample8_S8_L001_I1_001.fastq.gz,fastq fastq fastq,37968081102.0,301333977.0,GSM4547702 r2,0:8 1:26 2:92,A:10580168599;C:8638562811;G:9024245345;T:9723132070;N:1972277,8,26,92,,10580168599,8638562811,9024245345,9723132070,1972277,SRX8330063,SRS6649550,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95282,,0.06548,,0.8546,,0.52648,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59198,SRR11777190,SRX8330063,SRS6649550,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 3,GSM4547702,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547702,GSM4547702: dominant 3; Danio rerio; RNA Seq,GSM4547702,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547702,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample8 S8 L002 I1 001.fastq.gz   read2PairFiles=sample8 S8 L002 R1 001.fastq.gz   read3PairFiles=sample8 S8 L002 R2 001.fastq.gz,sample8_S8_L002_R2_001.fastq.gz sample8_S8_L002_R1_001.fastq.gz sample8_S8_L002_I1_001.fastq.gz,fastq fastq fastq,37640109906.0,298731031.0,GSM4547702 r3,0:8 1:26 2:92,A:10490817242;C:8564221041;G:8946179351;T:9637119181;N:1773091,8,26,92,,10490817242,8564221041,8946179351,9637119181,1773091,SRX8330063,SRS6649550,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95231,,0.06664,,0.85326,,0.52977,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59199,SRR11777185,SRX8330062,SRS6649549,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 2,GSM4547701,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547701,GSM4547701: dominant 2; Danio rerio; RNA Seq,GSM4547701,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547701,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample7 I1.fastq.gz   read2PairFiles=Next sample7 R1.fastq.gz   read3PairFiles=Next sample7 R2.fastq.gz,Next_sample7_R2.fastq.gz Next_sample7_R1.fastq.gz Next_sample7_I1.fastq.gz,fastq fastq fastq,8529279244.0,92709557.0,GSM4547701 r1,0:8 1:26 2:58,A:2302811456;C:1921371420;G:2078302909;T:2219976032;N:6817427,8,26,58,,2302811456,1921371420,2078302909,2219976032,6817427,SRX8330062,SRS6649549,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.94004,,0.09046,,0.85555,,0.50147,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59200,SRR11777186,SRX8330062,SRS6649549,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 2,GSM4547701,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547701,GSM4547701: dominant 2; Danio rerio; RNA Seq,GSM4547701,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547701,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample7 S7 L001 I1 001.fastq.gz   read2PairFiles=sample7 S7 L001 R1 001.fastq.gz   read3PairFiles=sample7 S7 L001 R2 001.fastq.gz,sample7_S7_L001_R2_001.fastq.gz sample7_S7_L001_R1_001.fastq.gz sample7_S7_L001_I1_001.fastq.gz,fastq fastq fastq,34725493026.0,275599151.0,GSM4547701 r2,0:8 1:26 2:92,A:9769026986;C:7706289537;G:8194381066;T:9053981896;N:1813541,8,26,92,,9769026986,7706289537,8194381066,9053981896,1813541,SRX8330062,SRS6649549,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.9448,,0.0817,,0.85336,,0.52553,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59201,SRR11777187,SRX8330062,SRS6649549,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 2,GSM4547701,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547701,GSM4547701: dominant 2; Danio rerio; RNA Seq,GSM4547701,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547701,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample7 S7 L002 I1 001.fastq.gz   read2PairFiles=sample7 S7 L002 R1 001.fastq.gz   read3PairFiles=sample7 S7 L002 R2 001.fastq.gz,sample7_S7_L002_R2_001.fastq.gz sample7_S7_L002_R1_001.fastq.gz sample7_S7_L002_I1_001.fastq.gz,fastq fastq fastq,34468767648.0,273561648.0,GSM4547701 r3,0:8 1:26 2:92,A:9700179724;C:7649330815;G:8132966012;T:8984661489;N:1629608,8,26,92,,9700179724,7649330815,8132966012,8984661489,1629608,SRX8330062,SRS6649549,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.94359,,0.08201,,0.85206,,0.52394,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59202,SRR11777182,SRX8330061,SRS6649548,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 1,GSM4547700,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547700,GSM4547700: dominant 1; Danio rerio; RNA Seq,GSM4547700,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547700,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample6 I1.fastq.gz   read2PairFiles=Next sample6 R1.fastq.gz   read3PairFiles=Next sample6 R2.fastq.gz,Next_sample6_R2.fastq.gz Next_sample6_R1.fastq.gz Next_sample6_I1.fastq.gz,fastq fastq fastq,7186504192.0,78114176.0,GSM4547700 r1,0:8 1:26 2:58,A:1890231359;C:1687648873;G:1767027837;T:1835889658;N:5706465,8,26,58,,1890231359,1687648873,1767027837,1835889658,5706465,SRX8330061,SRS6649548,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95103,,0.08633,,0.84997,,0.5111,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59203,SRR11777183,SRX8330061,SRS6649548,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 1,GSM4547700,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547700,GSM4547700: dominant 1; Danio rerio; RNA Seq,GSM4547700,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547700,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample6 S6 L001 I1 001.fastq.gz   read2PairFiles=sample6 S6 L001 R1 001.fastq.gz   read3PairFiles=sample6 S6 L001 R2 001.fastq.gz,sample6_S6_L001_R2_001.fastq.gz sample6_S6_L001_R1_001.fastq.gz sample6_S6_L001_I1_001.fastq.gz,fastq fastq fastq,35787988152.0,284031652.0,GSM4547700 r2,0:8 1:26 2:92,A:9781991861;C:8320736351;G:8552750572;T:9130655372;N:1853996,8,26,92,,9781991861,8320736351,8552750572,9130655372,1853996,SRX8330061,SRS6649548,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95998,,0.0809,,0.84891,,0.53314,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59204,SRR11777184,SRX8330061,SRS6649548,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,dominant 1,GSM4547700,,tissue:dominant|cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,dominant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,dominant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:sorted single color kidney marrow cells|sorting:sorted|age:8 mpf|genotype/variation:dominant Zebrabow color,GSM4547700,GSM4547700: dominant 1; Danio rerio; RNA Seq,GSM4547700,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547700,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample6 S6 L002 I1 001.fastq.gz   read2PairFiles=sample6 S6 L002 R1 001.fastq.gz   read3PairFiles=sample6 S6 L002 R2 001.fastq.gz,sample6_S6_L002_R2_001.fastq.gz sample6_S6_L002_R1_001.fastq.gz sample6_S6_L002_I1_001.fastq.gz,fastq fastq fastq,35545836942.0,282109817.0,GSM4547700 r3,0:8 1:26 2:92,A:9719207479;C:8264195207;G:8494199037;T:9066565654;N:1669565,8,26,92,,9719207479,8264195207,8494199037,9066565654,1669565,SRX8330061,SRS6649548,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95974,,0.0796,,0.85036,,0.54185,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59205,SRR11777179,SRX8330060,SRS6649547,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 3,GSM4547699,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547699,GSM4547699: mutant 3; Danio rerio; RNA Seq,GSM4547699,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547699,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample5 I1.fastq.gz   read2PairFiles=Next sample5 R1.fastq.gz   read3PairFiles=Next sample5 R2.fastq.gz,Next_sample5_R2.fastq.gz Next_sample5_R1.fastq.gz Next_sample5_I1.fastq.gz,fastq fastq fastq,7160703436.0,77833733.0,GSM4547699 r1,0:8 1:26 2:58,A:1816363574;C:1765855914;G:1734024608;T:1838721304;N:5738036,8,26,58,,1816363574,1765855914,1734024608,1838721304,5738036,SRX8330060,SRS6649547,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95273,,0.06131,,0.85693,,0.50453,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59206,SRR11777180,SRX8330060,SRS6649547,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 3,GSM4547699,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547699,GSM4547699: mutant 3; Danio rerio; RNA Seq,GSM4547699,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547699,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample5 S5 L001 I1 001.fastq.gz   read2PairFiles=sample5 S5 L001 R1 001.fastq.gz   read3PairFiles=sample5 S5 L001 R2 001.fastq.gz,sample5_S5_L001_R2_001.fastq.gz sample5_S5_L001_R1_001.fastq.gz sample5_S5_L001_I1_001.fastq.gz,fastq fastq fastq,39409717914.0,312775539.0,GSM4547699 r2,0:8 1:26 2:92,A:10331447053;C:9658224477;G:9315034438;T:10102971653;N:2040293,8,26,92,,10331447053,9658224477,9315034438,10102971653,2040293,SRX8330060,SRS6649547,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96833,,0.05626,,0.85336,,0.50422,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59207,SRR11777181,SRX8330060,SRS6649547,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 3,GSM4547699,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 3,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547699,GSM4547699: mutant 3; Danio rerio; RNA Seq,GSM4547699,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547699,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample5 S5 L002 I1 001.fastq.gz   read2PairFiles=sample5 S5 L002 R1 001.fastq.gz   read3PairFiles=sample5 S5 L002 R2 001.fastq.gz,sample5_S5_L002_R2_001.fastq.gz sample5_S5_L002_R1_001.fastq.gz sample5_S5_L002_I1_001.fastq.gz,fastq fastq fastq,39156927138.0,310769263.0,GSM4547699 r3,0:8 1:26 2:92,A:10269040558;C:9596019625;G:9255122700;T:10034901230;N:1843025,8,26,92,,10269040558,9596019625,9255122700,10034901230,1843025,SRX8330060,SRS6649547,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96923,,0.05633,,0.85356,,0.5018,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59208,SRR11777176,SRX8330059,SRS6649546,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 2,GSM4547698,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547698,GSM4547698: mutant 2; Danio rerio; RNA Seq,GSM4547698,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547698,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample4 I1.fastq.gz   read2PairFiles=Next sample4 R1.fastq.gz   read3PairFiles=Next sample4 R2.fastq.gz,Next_sample4_R2.fastq.gz Next_sample4_R1.fastq.gz Next_sample4_I1.fastq.gz,fastq fastq fastq,6117699620.0,66496735.0,GSM4547698 r1,0:8 1:26 2:58,A:1585513100;C:1461263890;G:1493383278;T:1572628286;N:4911066,8,26,58,,1585513100,1461263890,1493383278,1572628286,4911066,SRX8330059,SRS6649546,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95122,,0.07021,,0.85638,,0.50527,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59209,SRR11777177,SRX8330059,SRS6649546,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 2,GSM4547698,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547698,GSM4547698: mutant 2; Danio rerio; RNA Seq,GSM4547698,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547698,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample4 S4 L001 I1 001.fastq.gz   read2PairFiles=sample4 S4 L001 R1 001.fastq.gz   read3PairFiles=sample4 S4 L001 R2 001.fastq.gz,sample4_S4_L001_R2_001.fastq.gz sample4_S4_L001_R1_001.fastq.gz sample4_S4_L001_I1_001.fastq.gz,fastq fastq fastq,34901689032.0,276997532.0,GSM4547698 r2,0:8 1:26 2:92,A:9403468365;C:8264174645;G:8233892651;T:8998340867;N:1812504,8,26,92,,9403468365,8264174645,8233892651,8998340867,1812504,SRX8330059,SRS6649546,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.9607,,0.06192,,0.85251,,0.5091,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59210,SRR11777178,SRX8330059,SRS6649546,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 2,GSM4547698,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547698,GSM4547698: mutant 2; Danio rerio; RNA Seq,GSM4547698,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547698,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample4 S4 L002 I1 001.fastq.gz   read2PairFiles=sample4 S4 L002 R1 001.fastq.gz   read3PairFiles=sample4 S4 L002 R2 001.fastq.gz,sample4_S4_L002_I1_001.fastq.gz sample4_S4_L002_R1_001.fastq.gz sample4_S4_L002_R2_001.fastq.gz,fastq fastq fastq,34667396694.0,275138069.0,GSM4547698 r3,0:8 1:26 2:92,A:9343712751;C:8208672454;G:8177795137;T:8935581933;N:1634419,8,26,92,,9343712751,8208672454,8177795137,8935581933,1634419,SRX8330059,SRS6649546,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95991,,0.06275,,0.85269,,0.51362,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59211,SRR11777173,SRX8330058,SRS6649545,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 1,GSM4547697,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547697,GSM4547697: mutant 1; Danio rerio; RNA Seq,GSM4547697,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547697,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample3 I1.fastq.gz   read2PairFiles=Next sample3 R1.fastq.gz   read3PairFiles=Next sample3 R2.fastq.gz,Next_sample3_R2.fastq.gz Next_sample3_R1.fastq.gz Next_sample3_I1.fastq.gz,fastq fastq fastq,6871405756.0,74689193.0,GSM4547697 r1,0:8 1:26 2:58,A:1758336575;C:1648937548;G:1652034299;T:1806605610;N:5491724,8,26,58,,1758336575,1648937548,1652034299,1806605610,5491724,SRX8330058,SRS6649545,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95127,,0.08969,,0.8478,,0.50938,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59212,SRR11777174,SRX8330058,SRS6649545,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 1,GSM4547697,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547697,GSM4547697: mutant 1; Danio rerio; RNA Seq,GSM4547697,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547697,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample3 S3 L001 I1 001.fastq.gz   read2PairFiles=sample3 S3 L001 R1 001.fastq.gz   read3PairFiles=sample3 S3 L001 R2 001.fastq.gz,sample3_S3_L001_R2_001.fastq.gz sample3_S3_L001_R1_001.fastq.gz sample3_S3_L001_I1_001.fastq.gz,fastq fastq fastq,37240131222.0,295556597.0,GSM4547697 r2,0:8 1:26 2:92,A:9746145692;C:8895600775;G:8808801890;T:9787657360;N:1925505,8,26,92,,9746145692,8895600775,8808801890,9787657360,1925505,SRX8330058,SRS6649545,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96592,,0.08198,,0.84642,,0.50173,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59213,SRR11777175,SRX8330058,SRS6649545,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,mutant 1,GSM4547697,,tissue:mutant|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,mutant 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,mutant,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:guide RNAs targeting 20 different genes and cas9 mRNA,GSM4547697,GSM4547697: mutant 1; Danio rerio; RNA Seq,GSM4547697,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547697,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample3 S3 L002 I1 001.fastq.gz   read2PairFiles=sample3 S3 L002 R1 001.fastq.gz   read3PairFiles=sample3 S3 L002 R2 001.fastq.gz,sample3_S3_L002_R2_001.fastq.gz sample3_S3_L002_R1_001.fastq.gz sample3_S3_L002_I1_001.fastq.gz,fastq fastq fastq,36987042078.0,293547953.0,GSM4547697 r3,0:8 1:26 2:92,A:9682800852;C:8835028777;G:8748767711;T:9718714620;N:1730118,8,26,92,,9682800852,8835028777,8748767711,9718714620,1730118,SRX8330058,SRS6649545,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96466,,0.08156,,0.84605,,0.5026,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59214,SRR11777170,SRX8330057,SRS6649544,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 2,GSM4547696,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547696,GSM4547696: control 2; Danio rerio; RNA Seq,GSM4547696,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547696,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample2 I1.fastq.gz   read2PairFiles=Next sample2 R1.fastq.gz   read3PairFiles=Next sample2 R2.fastq.gz,Next_sample2_R2.fastq.gz Next_sample2_R1.fastq.gz Next_sample2_I1.fastq.gz,fastq fastq fastq,6538688308.0,71072699.0,GSM4547696 r1,0:8 1:26 2:58,A:1703837705;C:1534065984;G:1616771717;T:1678792786;N:5220116,8,26,58,,1703837705,1534065984,1616771717,1678792786,5220116,SRX8330057,SRS6649544,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95552,,0.0991,,0.85178,,0.51494,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59215,SRR11777171,SRX8330057,SRS6649544,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 2,GSM4547696,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547696,GSM4547696: control 2; Danio rerio; RNA Seq,GSM4547696,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547696,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample2 S2 L001 I1 001.fastq.gz   read2PairFiles=sample2 S2 L001 R1 001.fastq.gz   read3PairFiles=sample2 S2 L001 R2 001.fastq.gz,sample2_S2_L001_R2_001.fastq.gz sample2_S2_L001_R1_001.fastq.gz sample2_S2_L001_I1_001.fastq.gz,fastq fastq fastq,40686172002.0,322906127.0,GSM4547696 r2,0:8 1:26 2:92,A:11105689093;C:9483757136;G:9622172382;T:10472439389;N:2114002,8,26,92,,11105689093,9483757136,9622172382,10472439389,2114002,SRX8330057,SRS6649544,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96005,,0.09149,,0.8479,,0.52442,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59216,SRR11777172,SRX8330057,SRS6649544,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 2,GSM4547696,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 2,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547696,GSM4547696: control 2; Danio rerio; RNA Seq,GSM4547696,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547696,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample2 S2 L002 I1 001.fastq.gz   read2PairFiles=sample2 S2 L002 R1 001.fastq.gz   read3PairFiles=sample2 S2 L002 R2 001.fastq.gz,sample2_S2_L002_I1_001.fastq.gz sample2_S2_L002_R1_001.fastq.gz sample2_S2_L002_R2_001.fastq.gz,fastq fastq fastq,40394060784.0,320587784.0,GSM4547696 r3,0:8 1:26 2:92,A:11030989263;C:9415697570;G:9551942377;T:10393536337;N:1895237,8,26,92,,11030989263,9415697570,9551942377,10393536337,1895237,SRX8330057,SRS6649544,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96032,,0.09188,,0.84571,,0.51147,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59217,SRR11777167,SRX8330056,SRS6649543,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 1,GSM4547695,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547695,GSM4547695: control 1; Danio rerio; RNA Seq,GSM4547695,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547695,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Next sample1 I1.fastq.gz   read2PairFiles=Next sample1 R1.fastq.gz   read3PairFiles=Next sample1 R2.fastq.gz,Next_sample1_I1.fastq.gz Next_sample1_R1.fastq.gz Next_sample1_R2.fastq.gz,fastq fastq fastq,6194482636.0,67331333.0,GSM4547695 r1,0:8 1:26 2:58,A:1570433644;C:1511080065;G:1496787496;T:1611232189;N:4949242,8,26,58,,1570433644,1511080065,1496787496,1611232189,4949242,SRX8330056,SRS6649543,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95006,,0.07448,,0.85563,,0.43909,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59218,SRR11777168,SRX8330056,SRS6649543,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 1,GSM4547695,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547695,GSM4547695: control 1; Danio rerio; RNA Seq,GSM4547695,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547695,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample1 S1 L001 I1 001.fastq.gz   read2PairFiles=sample1 S1 L001 R1 001.fastq.gz   read3PairFiles=sample1 S1 L001 R2 001.fastq.gz,sample1_S1_L001_I1_001.fastq.gz sample1_S1_L001_R1_001.fastq.gz sample1_S1_L001_R2_001.fastq.gz,fastq fastq fastq,42139701324.0,334442074.0,GSM4547695 r2,0:8 1:26 2:92,A:10979793082;C:10192056919;G:10036291604;T:10929392375;N:2167344,8,26,92,,10979793082,10192056919,10036291604,10929392375,2167344,SRX8330056,SRS6649543,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96205,,0.06669,,0.85234,,0.51114,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59219,SRR11777169,SRX8330056,SRS6649543,SRP261272,PRJNA631927,Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis [I],GSE150373,Transcriptome Analysis,Clonal hematopoiesis CH results from enhanced fitness of a mutant hematopoietic stem and progenitor cell HSPC  but how such clones expand is unclear. Here  we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH associated genes  like asxl1  promoted clonal dominance. Single cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator  nr4a1  abrogated the ability of asxl1 mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Overall design: To analyze the effect of multiplex CRISPR mutagenesis on zebrafish marrow cells  we injected Zebrabow zebrafish embryos with guide RNAs targeting 20 different genes and cas9 mRNA. Control clutchmates were injected with 2 control guide RNAs and cas9 mRNA. These fish were grown to maturity. Whole kidney marrow cells from 2 control and 3 mutant zebrafish were collected for preparation for 10x single cell RNA sequencing. A third set of samples were sorted kidney marrow cells of the dominant Zebrabow color from the same three mutant zebrafish.,parent bioproject:PRJNA734615,pubmed:34735227,,control 1,GSM4547695,,tissue:control|cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,control 1,The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene level counts were imported into R using the Seurat suite version 3.0 Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets  gene level count values were summed to retain a final count matrix of 35 227 features across 39 424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: 1 >= 500 UMIs per cell; 2 >= 200 genes per cell; 3 >= 0.7 novelty score  calculated as log10genes detected / log10UMI counts per cell; and 4 <= 10 percentage of reads mapping to mitochondrial genes. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms,control,No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 month,cell type:unsorted whole kidney marrow cells|sorting:unsorted|age:8 mpf|genotype/variation:2 control guide RNAs and cas9 mRNA,GSM4547695,GSM4547695: control 1; Danio rerio; RNA Seq,GSM4547695,,1,post euthanasia  the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin  placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40 μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA Gemini Bio Products  then washed again post spinning down at 350 x g for 5 minutes at 4°C and counted post resuspension in DPBS/BSA to target 10 000 cells per reaction. We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation single cell RNA sequencing  10x genomics,GEO Accession:GSM4547695,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP261272,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=sample1 S1 L002 I1 001.fastq.gz   read2PairFiles=sample1 S1 L002 R1 001.fastq.gz   read3PairFiles=sample1 S1 L002 R2 001.fastq.gz,sample1_S1_L002_R2_001.fastq.gz sample1_S1_L002_R1_001.fastq.gz sample1_S1_L002_I1_001.fastq.gz,fastq fastq fastq,41867006580.0,332277830.0,GSM4547695 r3,0:8 1:26 2:92,A:10912078962;C:10125686513;G:9971217295;T:10856057042;N:1966768,8,26,92,,10912078962,10125686513,9971217295,10856057042,1966768,SRX8330056,SRS6649543,SRA1074695,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.96295,,0.06599,,0.85299,,0.51294,,92,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-05-12,Adult,Adult,Multi-tissue,Multi-system
59870,SRR12055985,SRX8584378,SRS6875885,SRP268040,PRJNA640655,Emx2 defines bidirectional polarity of neuromasts by changing hair bundle orientation and not hair cell positions,GSE152859,Other,The bidirectional sensitivity of neuromasts to water flow in the zebrafish lateral line system is attributed to the opposite orientation of the hair bundles on top of sensory hair cells HC within a neuromast. post each HC precursor divides to form two nascent HCs  HCs of the same bundle orientation are positioned on one side of the neuromast  across from HCs with opposite bundle orientation. The transcription factor emx2 is expressed in only one of the sibling HCs. Loss or gain of function of emx2 in HCs causes unidirectional hair bundle orientation in neuromasts. It is not clear whether Emx2 is required specifically in establishing hair bundle orientation post HC formation or Emx2 has an earlier role in HC fate and/or positioning  which indirectly changes hair bundle orientation. A phenomenon  in which nascent sibling HCs exchange positions with each other  has been postulated to be the mechanism for HCs to acquire their designated positions within the neuromast. We asked whether Emx2 is involved in positional acquisition of HCs within the neuromast. Using live imaging and our emx2 reporter we find that the HC rearrangement  redefined as two processes named Rock & Roll are required for HCs to acquire their positions. Although Emx2 regulates the duration of the Rock and the frequency of Roll of nascent HCs  it is not required for their positional acquisition. Instead  Emx2 regulates the morphology of nascent HCs  which facilitates the rearrangement process. Overall design: Single cell RNA sequencing on FACS sorted Hair Cells from zebrafish neuromast in 4dpf wild type larvae.,,pubmed:33377867,,scRNA seq zebrafish 4dpf  neuromast hair cells,GSM4628143,,tissue:P4 zebrafish hair cell|genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,scRNA seq zebrafish 4dpf  neuromast hair cells,Basecalls performed using Illumina’s RTA 2.0.12. Sample demultiplexing  alignment and gene counts were performed using the 10X Genomics CellRanger v3.0.1 pipeline using default settings. Secondary analysis was performed using SCANPY version 1.4.4. Genome build: not provided Supplementary files format and content: barcodes.tsv.gz: cell barcodes Supplementary files format and content: features.tsv.gz: gene names Supplementary files format and content: matrix.mtx.gz: expression matrix Supplementary files format and content: filtered feature bc matrix.h5: expression matrix in HDF5 format,P4 zebrafish hair cell,n/a,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer’s instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,n/a,genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,GSM4628143,GSM4628143: scRNA seq zebrafish 4dpf  neuromast hair cells; Danio rerio; RNA Seq,GSM4628143,,1,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer's instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,GEO Accession:GSM4628143,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP268040,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=ZM4 S1 L001 I1 001.fastq.gz   read2PairFiles=ZM4 S1 L001 R1 001.fastq.gz   read3PairFiles=ZM4 S1 L001 R2 001.fastq.gz,ZM4_S1_L001_I1_001.fastq.gz ZM4_S1_L001_R1_001.fastq.gz ZM4_S1_L001_R2_001.fastq.gz,fastq fastq fastq,5422224324.0,41077457.0,GSM4628143 r1,0:8 1:26 2:98,A:1539837924;C:1167111399;G:1177239259;T:1536971308;N:1064434,8,26,98,,1539837924,1167111399,1177239259,1536971308,1064434,SRX8584378,SRS6875885,SRA1089164,GEO,"Molecular Biology, NIDCD",1,0.93866,,0.12434,,0.8229,,0.42198,,98,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Larval,Larval,Multi-tissue,Multi-system
59871,SRR12055986,SRX8584378,SRS6875885,SRP268040,PRJNA640655,Emx2 defines bidirectional polarity of neuromasts by changing hair bundle orientation and not hair cell positions,GSE152859,Other,The bidirectional sensitivity of neuromasts to water flow in the zebrafish lateral line system is attributed to the opposite orientation of the hair bundles on top of sensory hair cells HC within a neuromast. post each HC precursor divides to form two nascent HCs  HCs of the same bundle orientation are positioned on one side of the neuromast  across from HCs with opposite bundle orientation. The transcription factor emx2 is expressed in only one of the sibling HCs. Loss or gain of function of emx2 in HCs causes unidirectional hair bundle orientation in neuromasts. It is not clear whether Emx2 is required specifically in establishing hair bundle orientation post HC formation or Emx2 has an earlier role in HC fate and/or positioning  which indirectly changes hair bundle orientation. A phenomenon  in which nascent sibling HCs exchange positions with each other  has been postulated to be the mechanism for HCs to acquire their designated positions within the neuromast. We asked whether Emx2 is involved in positional acquisition of HCs within the neuromast. Using live imaging and our emx2 reporter we find that the HC rearrangement  redefined as two processes named Rock & Roll are required for HCs to acquire their positions. Although Emx2 regulates the duration of the Rock and the frequency of Roll of nascent HCs  it is not required for their positional acquisition. Instead  Emx2 regulates the morphology of nascent HCs  which facilitates the rearrangement process. Overall design: Single cell RNA sequencing on FACS sorted Hair Cells from zebrafish neuromast in 4dpf wild type larvae.,,pubmed:33377867,,scRNA seq zebrafish 4dpf  neuromast hair cells,GSM4628143,,tissue:P4 zebrafish hair cell|genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,scRNA seq zebrafish 4dpf  neuromast hair cells,Basecalls performed using Illumina’s RTA 2.0.12. Sample demultiplexing  alignment and gene counts were performed using the 10X Genomics CellRanger v3.0.1 pipeline using default settings. Secondary analysis was performed using SCANPY version 1.4.4. Genome build: not provided Supplementary files format and content: barcodes.tsv.gz: cell barcodes Supplementary files format and content: features.tsv.gz: gene names Supplementary files format and content: matrix.mtx.gz: expression matrix Supplementary files format and content: filtered feature bc matrix.h5: expression matrix in HDF5 format,P4 zebrafish hair cell,n/a,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer’s instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,n/a,genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,GSM4628143,GSM4628143: scRNA seq zebrafish 4dpf  neuromast hair cells; Danio rerio; RNA Seq,GSM4628143,,1,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer's instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,GEO Accession:GSM4628143,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP268040,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=ZM4 S1 L002 I1 001.fastq.gz   read2PairFiles=ZM4 S1 L002 R1 001.fastq.gz   read3PairFiles=ZM4 S1 L002 R2 001.fastq.gz,ZM4_S1_L002_I1_001.fastq.gz ZM4_S1_L002_R1_001.fastq.gz ZM4_S1_L002_R2_001.fastq.gz,fastq fastq fastq,5480072136.0,41515698.0,GSM4628143 r2,0:8 1:26 2:98,A:1556115839;C:1180006462;G:1189325251;T:1553616619;N:1007965,8,26,98,,1556115839,1180006462,1189325251,1553616619,1007965,SRX8584378,SRS6875885,SRA1089164,GEO,"Molecular Biology, NIDCD",1,0.93811,,0.12465,,0.8227,,0.42538,,98,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Larval,Larval,Multi-tissue,Multi-system
59872,SRR12055987,SRX8584378,SRS6875885,SRP268040,PRJNA640655,Emx2 defines bidirectional polarity of neuromasts by changing hair bundle orientation and not hair cell positions,GSE152859,Other,The bidirectional sensitivity of neuromasts to water flow in the zebrafish lateral line system is attributed to the opposite orientation of the hair bundles on top of sensory hair cells HC within a neuromast. post each HC precursor divides to form two nascent HCs  HCs of the same bundle orientation are positioned on one side of the neuromast  across from HCs with opposite bundle orientation. The transcription factor emx2 is expressed in only one of the sibling HCs. Loss or gain of function of emx2 in HCs causes unidirectional hair bundle orientation in neuromasts. It is not clear whether Emx2 is required specifically in establishing hair bundle orientation post HC formation or Emx2 has an earlier role in HC fate and/or positioning  which indirectly changes hair bundle orientation. A phenomenon  in which nascent sibling HCs exchange positions with each other  has been postulated to be the mechanism for HCs to acquire their designated positions within the neuromast. We asked whether Emx2 is involved in positional acquisition of HCs within the neuromast. Using live imaging and our emx2 reporter we find that the HC rearrangement  redefined as two processes named Rock & Roll are required for HCs to acquire their positions. Although Emx2 regulates the duration of the Rock and the frequency of Roll of nascent HCs  it is not required for their positional acquisition. Instead  Emx2 regulates the morphology of nascent HCs  which facilitates the rearrangement process. Overall design: Single cell RNA sequencing on FACS sorted Hair Cells from zebrafish neuromast in 4dpf wild type larvae.,,pubmed:33377867,,scRNA seq zebrafish 4dpf  neuromast hair cells,GSM4628143,,tissue:P4 zebrafish hair cell|genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,scRNA seq zebrafish 4dpf  neuromast hair cells,Basecalls performed using Illumina’s RTA 2.0.12. Sample demultiplexing  alignment and gene counts were performed using the 10X Genomics CellRanger v3.0.1 pipeline using default settings. Secondary analysis was performed using SCANPY version 1.4.4. Genome build: not provided Supplementary files format and content: barcodes.tsv.gz: cell barcodes Supplementary files format and content: features.tsv.gz: gene names Supplementary files format and content: matrix.mtx.gz: expression matrix Supplementary files format and content: filtered feature bc matrix.h5: expression matrix in HDF5 format,P4 zebrafish hair cell,n/a,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer’s instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,n/a,genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,GSM4628143,GSM4628143: scRNA seq zebrafish 4dpf  neuromast hair cells; Danio rerio; RNA Seq,GSM4628143,,1,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer's instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,GEO Accession:GSM4628143,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP268040,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=ZM4 S1 L003 I1 001.fastq.gz   read2PairFiles=ZM4 S1 L003 R1 001.fastq.gz   read3PairFiles=ZM4 S1 L003 R2 001.fastq.gz,ZM4_S1_L003_I1_001.fastq.gz ZM4_S1_L003_R1_001.fastq.gz ZM4_S1_L003_R2_001.fastq.gz,fastq fastq fastq,5589739452.0,42346511.0,GSM4628143 r3,0:8 1:26 2:98,A:1586134828;C:1202961360;G:1215543516;T:1584245728;N:854020,8,26,98,,1586134828,1202961360,1215543516,1584245728,854020,SRX8584378,SRS6875885,SRA1089164,GEO,"Molecular Biology, NIDCD",1,0.93749,,0.12195,,0.82286,,0.41205,,98,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Larval,Larval,Multi-tissue,Multi-system
59873,SRR12055988,SRX8584378,SRS6875885,SRP268040,PRJNA640655,Emx2 defines bidirectional polarity of neuromasts by changing hair bundle orientation and not hair cell positions,GSE152859,Other,The bidirectional sensitivity of neuromasts to water flow in the zebrafish lateral line system is attributed to the opposite orientation of the hair bundles on top of sensory hair cells HC within a neuromast. post each HC precursor divides to form two nascent HCs  HCs of the same bundle orientation are positioned on one side of the neuromast  across from HCs with opposite bundle orientation. The transcription factor emx2 is expressed in only one of the sibling HCs. Loss or gain of function of emx2 in HCs causes unidirectional hair bundle orientation in neuromasts. It is not clear whether Emx2 is required specifically in establishing hair bundle orientation post HC formation or Emx2 has an earlier role in HC fate and/or positioning  which indirectly changes hair bundle orientation. A phenomenon  in which nascent sibling HCs exchange positions with each other  has been postulated to be the mechanism for HCs to acquire their designated positions within the neuromast. We asked whether Emx2 is involved in positional acquisition of HCs within the neuromast. Using live imaging and our emx2 reporter we find that the HC rearrangement  redefined as two processes named Rock & Roll are required for HCs to acquire their positions. Although Emx2 regulates the duration of the Rock and the frequency of Roll of nascent HCs  it is not required for their positional acquisition. Instead  Emx2 regulates the morphology of nascent HCs  which facilitates the rearrangement process. Overall design: Single cell RNA sequencing on FACS sorted Hair Cells from zebrafish neuromast in 4dpf wild type larvae.,,pubmed:33377867,,scRNA seq zebrafish 4dpf  neuromast hair cells,GSM4628143,,tissue:P4 zebrafish hair cell|genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,scRNA seq zebrafish 4dpf  neuromast hair cells,Basecalls performed using Illumina’s RTA 2.0.12. Sample demultiplexing  alignment and gene counts were performed using the 10X Genomics CellRanger v3.0.1 pipeline using default settings. Secondary analysis was performed using SCANPY version 1.4.4. Genome build: not provided Supplementary files format and content: barcodes.tsv.gz: cell barcodes Supplementary files format and content: features.tsv.gz: gene names Supplementary files format and content: matrix.mtx.gz: expression matrix Supplementary files format and content: filtered feature bc matrix.h5: expression matrix in HDF5 format,P4 zebrafish hair cell,n/a,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer’s instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,n/a,genotype/variation:Tgmyo6b:RiboTag|developmental stage:4 dpf|cell type:Neuromast hair cells,GSM4628143,GSM4628143: scRNA seq zebrafish 4dpf  neuromast hair cells; Danio rerio; RNA Seq,GSM4628143,,1,Zebrafish larvae Tgmyo6b: RiboTag at 4 dpf were dissected to remove the head  which included the ear. Approximately 400 dissected bodies in batches of 50 were dissociated by adding 0.5 ml of 0.5% trypsin Gibco  cat# 15400054 and triturated with 1 ml pipette tip for 30 seconds at 30°C. The dissociated cells were filtered with 40 μm strainer Pluriselect  cat# 43 50040 50 and washed with PBS twice before adding DMEM containing 10% FBS and spun down by centrifugation at 700g rpm for 5 min at 4°C. Cells were resuspended and kept in DMEM before processing for FACS. scRNA seq was carried out with 10X Genomics single cell platform 10X Genomics  Pleasanton  CA. USA. Approximately 5000 live cells post FACS in a maximum volume of 34 microliters were loaded on a 10X Genomics Chromium Controller 10x Genomics. Chromium Single Cell three prime Library and Gel Bead Kit v2 10X Genomics was used for libraries preparation according to manufacturer's instructions. Quality of the libraries was evaluated on a Fragment Analyzer instrument Agilent and sequenced on a NextSeq 500 sequencer Illumina with the following paired read lengths: 26 bp Read 1  8 bp I7 Index and 98 bp Read 2.,GEO Accession:GSM4628143,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP268040,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=ZM4 S1 L004 I1 001.fastq.gz   read2PairFiles=ZM4 S1 L004 R1 001.fastq.gz   read3PairFiles=ZM4 S1 L004 R2 001.fastq.gz,ZM4_S1_L004_I1_001.fastq.gz ZM4_S1_L004_R1_001.fastq.gz ZM4_S1_L004_R2_001.fastq.gz,fastq fastq fastq,5495099808.0,41629544.0,GSM4628143 r4,0:8 1:26 2:98,A:1559732557;C:1183298748;G:1193530338;T:1557803123;N:735042,8,26,98,,1559732557,1183298748,1193530338,1557803123,735042,SRX8584378,SRS6875885,SRA1089164,GEO,"Molecular Biology, NIDCD",1,0.93891,,0.12244,,0.82181,,0.42894,,98,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Larval,Larval,Multi-tissue,Multi-system
62679,SRR13311744,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_1_S21_L001_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_1_S21_L001_R2_001.fastq.gz,fastq fastq,23474658448.0,77935654.0,GSM4990514 r1,0:150.58 1:150.62,A:5315661572;C:4260464374;G:7908155075;T:5989858000;N:519427,150,150,,,5315661572,4260464374,7908155075,5989858000,519427,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90618,0.0,0.11433,1.0,0.80178,,0.51559,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62680,SRR13311745,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_1_S21_L002_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_1_S21_L002_R2_001.fastq.gz,fastq fastq,23453047207.0,77864343.0,GSM4990514 r2,0:150.58 1:150.62,A:5353428042;C:4272250712;G:7801502473;T:6025426926;N:439054,150,150,,,5353428042,4272250712,7801502473,6025426926,439054,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90571,0.0,0.11445,1.0,0.80326,,0.51014,149,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62681,SRR13311746,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_2_S22_L001_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_2_S22_L001_R2_001.fastq.gz,fastq fastq,18693961077.0,62064026.0,GSM4990514 r3,0:150.58 1:150.62,A:4223627306;C:3394276969;G:6298948837;T:4776692177;N:415788,150,150,,,4223627306,3394276969,6298948837,4776692177,415788,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90754,0.0,0.11555,1.0,0.8018,,0.50793,150,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62682,SRR13311747,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_2_S22_L002_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_2_S22_L002_R2_001.fastq.gz,fastq fastq,18650610191.0,61920476.0,GSM4990514 r4,0:150.58 1:150.62,A:4248232322;C:3398942457;G:6205002760;T:4798080435;N:352217,150,150,,,4248232322,3398942457,6205002760,4798080435,352217,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90621,0.0,0.11451,1.0,0.80223,,0.51751,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62683,SRR13311748,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_3_S23_L001_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_3_S23_L001_R2_001.fastq.gz,fastq fastq,21139583045.0,70183976.0,GSM4990514 r5,0:150.58 1:150.62,A:4766130433;C:3847270802;G:7123755600;T:5401957528;N:468682,150,150,,,4766130433,3847270802,7123755600,5401957528,468682,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90939,0.0,0.11373,1.0,0.80044,,0.51922,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62684,SRR13311749,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_3_S23_L002_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_3_S23_L002_R2_001.fastq.gz,fastq fastq,21127496209.0,70144256.0,GSM4990514 r6,0:150.58 1:150.62,A:4801779342;C:3857990471;G:7030522815;T:5436808841;N:394740,150,150,,,4801779342,3857990471,7030522815,5436808841,394740,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90941,0.0,0.11435,1.0,0.80087,,0.51663,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62685,SRR13311750,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_4_S24_L001_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_4_S24_L001_R2_001.fastq.gz,fastq fastq,19213599440.0,63789538.0,GSM4990514 r7,0:150.58 1:150.62,A:4332795032;C:3494053982;G:6477323231;T:4908999176;N:428019,150,150,,,4332795032,3494053982,6477323231,4908999176,428019,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.9086,0.0,0.11426,1.0,0.80257,,0.51017,151,150,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62686,SRR13311751,SRX9739651,SRS7936985,SRP299375,PRJNA688157,Single cell RNA analysis of trunk neural crest cells in zebrafish,GSE163907,Other,Single cell RNA seq data of tgsox10:GFP cells from 20 hpf 24 hpf zebrafish. Sequenced with 10x genomics. Overall design: tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,pubmed:34397384,,tgsox10:GFP,GSM4990514,,source name:zebrafish trunk tgsox10:GFP positive cells|genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,tgsox10:GFP,cellranger v3.2 seurat v3.2.2 Genome build: GRCz11 Supplementary files format and content: features  barcodes  counts matrix for scRNAseq Supplementary files format and content: Cluster assignments,zebrafish trunk tgsox10:GFP positive cells,,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,,genotype:tgsox10:GFP ba2|tissue:trunk neural crest cells|developmental stage:24 hpf,GSM4990514,GSM4990514: tgsox10:GFP; Danio rerio; RNA Seq,GSM4990514,,1,tgsox10:GFP cells were isolated by FACS from dissociated trunks of 20 24hpf zebrafish and submitted to 10x Sequencing,GEO Accession:GSM4990514,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP299375,,,Cut_Sox10_GFP_24hpf_12142018_4_S24_L002_R1_001.fastq.gz Cut_Sox10_GFP_24hpf_12142018_4_S24_L002_R2_001.fastq.gz,fastq fastq,19195135075.0,63728553.0,GSM4990514 r8,0:150.58 1:150.62,A:4363501238;C:3503247240;G:6390188740;T:4937837783;N:360074,150,150,,,4363501238,3503247240,6390188740,4937837783,360074,SRX9739651,SRS7936985,SRA1178122,GEO,"Artinger Laboratory, Craniofacial Biology, University of Colorado Denver | Anschutz",2,0.0,0.90626,0.0,0.11537,1.0,0.8032,,0.50845,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-12-28,Pharyngula,Embryo,Multi-tissue,Multi-system
62904,SRR13429041,SRX9843973,SRS8025681,SRP301615,PRJNA691919,A single cell transcriptomic atlas identifies synapse associated microglia in the developing zebrafish single cells,GSE164772,Transcriptome Analysis,This study profiles developing and adult zebrafish macrophages and defines regional and functional microglial subtypes. Overall design: Macrophages Mpeg+ and CD45+ cells were sorted and sequenced  from 28 dpf and 6 mpf zebrafish,,pubmed:34625548,,Adult A,GSM5018536,,tissue:Brain and Spinal Cord Mpeg+ and CD45+|age:6 mpf A|cell type:Mpeg+ myeloid cells and CD45+ immune cells,Adult A,"Cell Ranger 3.1.0 STAR alignment with Danio rerio zebrafish transcriptome z11 Quality control to select cells with <10% mitochondrial RNA  500 3 000 unique genes  and 1 200 15 000 unique mRNA counts [scaledata only] Log normalization Seurat v3.2 with scale factor 10 000 [scaledata only] most variable genes scaled with ""vst"" algorithm and #genes/cell regressed out Seurat v3.2 Genome build: z11 Supplementary files format and content: Counts: unnormalized count data for all cells included in the manuscript Supplementary files format and content: Metadata: file containing age  individual  and cluster assignment for each cell Supplementary files format and content: sc dr Silva2021Counts.txt: All cells in paper Supplementary files format and content: sc dr Silva2021 metadata.csv: All cells in paper Supplementary files format and content: sc dr Silva2021 JMscaledata.txt: Juvenile Mpeg+ cells Supplementary files format and content: sc dr Silva2021 Jscaledata.txt: All Juvenile cells Supplementary files format and content: sc dr Silva2021 Ascaledata.txt: Adult and Juvenile Mpeg+ cells",Brain and Spinal Cord Mpeg+ and CD45+,,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,,age:6 mpf A|cell type:Mpeg+ myeloid cells and CD45+ immune cells,GSM5018536,GSM5018536: Adult A; Danio rerio; RNA Seq,GSM5018536,,1,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,GEO Accession:GSM5018536,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP301615,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Sample3  A1  S2 L001 I1 001.fastq.gz   read2PairFiles=Sample3  A1  S2 L001 R1 001.fastq.gz   read3PairFiles=Sample3  A1  S2 L001 R2 001.fastq.gz,Sample3-_A1__S2_L001_I1_001.fastq.gz Sample3-_A1__S2_L001_R1_001.fastq.gz Sample3-_A1__S2_L001_R2_001.fastq.gz,fastq fastq fastq,5863809770.0,45106229.0,GSM5018536 r1,0:8 1:28 2:94,A:1686208936;C:1248351000;G:1297163723;T:1629033266;N:3052845,8,28,94,,1686208936,1248351000,1297163723,1629033266,3052845,SRX9843973,SRS8025681,SRA1183553,GEO,"Anna Molofsky Lab, Psychiatry, UCSF",1,0.89063,,0.3045,,0.79632,,0.57216,,94,,B,,usable mapping rate,illumina,novaseq_era,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2021-01-13,Adult,Adult,Multi-tissue,Multi-system
62905,SRR13429040,SRX9843972,SRS8025678,SRP301615,PRJNA691919,A single cell transcriptomic atlas identifies synapse associated microglia in the developing zebrafish single cells,GSE164772,Transcriptome Analysis,This study profiles developing and adult zebrafish macrophages and defines regional and functional microglial subtypes. Overall design: Macrophages Mpeg+ and CD45+ cells were sorted and sequenced  from 28 dpf and 6 mpf zebrafish,,pubmed:34625548,,Juvenile B,GSM5018535,,tissue:Brain and Spinal Cord Mpeg+ and CD45+|age:28 dpf B|cell type:Mpeg+ myeloid cells and CD45+ immune cells,Juvenile B,"Cell Ranger 3.1.0 STAR alignment with Danio rerio zebrafish transcriptome z11 Quality control to select cells with <10% mitochondrial RNA  500 3 000 unique genes  and 1 200 15 000 unique mRNA counts [scaledata only] Log normalization Seurat v3.2 with scale factor 10 000 [scaledata only] most variable genes scaled with ""vst"" algorithm and #genes/cell regressed out Seurat v3.2 Genome build: z11 Supplementary files format and content: Counts: unnormalized count data for all cells included in the manuscript Supplementary files format and content: Metadata: file containing age  individual  and cluster assignment for each cell Supplementary files format and content: sc dr Silva2021Counts.txt: All cells in paper Supplementary files format and content: sc dr Silva2021 metadata.csv: All cells in paper Supplementary files format and content: sc dr Silva2021 JMscaledata.txt: Juvenile Mpeg+ cells Supplementary files format and content: sc dr Silva2021 Jscaledata.txt: All Juvenile cells Supplementary files format and content: sc dr Silva2021 Ascaledata.txt: Adult and Juvenile Mpeg+ cells",Brain and Spinal Cord Mpeg+ and CD45+,,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,,age:28 dpf B|cell type:Mpeg+ myeloid cells and CD45+ immune cells,GSM5018535,GSM5018535: Juvenile B; Danio rerio; RNA Seq,GSM5018535,,1,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,GEO Accession:GSM5018535,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP301615,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Sample2  L2  S4 L001 I1 001.fastq.gz   read2PairFiles=Sample2  L2  S4 L001 R1 001.fastq.gz   read3PairFiles=Sample2  L2  S4 L001 R2 001.fastq.gz,Sample2-_L2__S4_L001_I1_001.fastq.gz Sample2-_L2__S4_L001_R1_001.fastq.gz Sample2-_L2__S4_L001_R2_001.fastq.gz,fastq fastq fastq,20625538440.0,158657988.0,GSM5018535 r1,0:8 1:28 2:94,A:5854974563;C:4547250454;G:4552388689;T:5660267648;N:10657086,8,28,94,,5854974563,4547250454,4552388689,5660267648,10657086,SRX9843972,SRS8025678,SRA1183553,GEO,"Anna Molofsky Lab, Psychiatry, UCSF",1,0.90631,,0.26392,,0.78407,,0.56464,,94,,B,,usable mapping rate,illumina,novaseq_era,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2021-01-13,Larval,Larval,Multi-tissue,Multi-system
62906,SRR13429039,SRX9843971,SRS8025680,SRP301615,PRJNA691919,A single cell transcriptomic atlas identifies synapse associated microglia in the developing zebrafish single cells,GSE164772,Transcriptome Analysis,This study profiles developing and adult zebrafish macrophages and defines regional and functional microglial subtypes. Overall design: Macrophages Mpeg+ and CD45+ cells were sorted and sequenced  from 28 dpf and 6 mpf zebrafish,,pubmed:34625548,,Juvenile A,GSM5018534,,tissue:Brain and Spinal Cord Mpeg+ and CD45+|age:28 dpf A|cell type:Mpeg+ myeloid cells and CD45+ immune cells,Juvenile A,"Cell Ranger 3.1.0 STAR alignment with Danio rerio zebrafish transcriptome z11 Quality control to select cells with <10% mitochondrial RNA  500 3 000 unique genes  and 1 200 15 000 unique mRNA counts [scaledata only] Log normalization Seurat v3.2 with scale factor 10 000 [scaledata only] most variable genes scaled with ""vst"" algorithm and #genes/cell regressed out Seurat v3.2 Genome build: z11 Supplementary files format and content: Counts: unnormalized count data for all cells included in the manuscript Supplementary files format and content: Metadata: file containing age  individual  and cluster assignment for each cell Supplementary files format and content: sc dr Silva2021Counts.txt: All cells in paper Supplementary files format and content: sc dr Silva2021 metadata.csv: All cells in paper Supplementary files format and content: sc dr Silva2021 JMscaledata.txt: Juvenile Mpeg+ cells Supplementary files format and content: sc dr Silva2021 Jscaledata.txt: All Juvenile cells Supplementary files format and content: sc dr Silva2021 Ascaledata.txt: Adult and Juvenile Mpeg+ cells",Brain and Spinal Cord Mpeg+ and CD45+,,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,,age:28 dpf A|cell type:Mpeg+ myeloid cells and CD45+ immune cells,GSM5018534,GSM5018534: Juvenile A; Danio rerio; RNA Seq,GSM5018534,,1,Zebrafish brains from mpeg:GFP; CD45:DsRed fish were homogenized and sorted to isolate single CD45:DsRed+ and double mpeg:GFP+/CD45:DsRed+ positive cells 10x Genomics v3.1 three prime Single Index Chip Kit B was used to make a single cell suspension in gel beads  and the v3 three prime Library Kit was used to prepare cDNA,GEO Accession:GSM5018534,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP301615,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=Sample1  L1  S1 L001 I1 001.fastq.gz   read2PairFiles=Sample1  L1  S1 L001 R1 001.fastq.gz   read3PairFiles=Sample1  L1  S1 L001 R2 001.fastq.gz,Sample1-_L1__S1_L001_I1_001.fastq.gz Sample1-_L1__S1_L001_R1_001.fastq.gz Sample1-_L1__S1_L001_R2_001.fastq.gz,fastq fastq fastq,12770179370.0,98232149.0,GSM5018534 r1,0:8 1:28 2:94,A:3657439948;C:2724528296;G:2851650695;T:3529904486;N:6655945,8,28,94,,3657439948,2724528296,2851650695,3529904486,6655945,SRX9843971,SRS8025680,SRA1183553,GEO,"Anna Molofsky Lab, Psychiatry, UCSF",1,0.89472,,0.26792,,0.78837,,0.55341,,94,,B,,usable mapping rate,illumina,novaseq_era,unknown,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2021-01-13,Larval,Larval,Multi-tissue,Multi-system
63226,SRR13660516,SRX10052201,SRS8215327,SRP305389,PRJNA700672,Endothelial progenitor migration and differentiation is regulated by distinct transcriptional effectors of Cloche/Npas4l,GSE166396,Transcriptome Analysis,Embryonic cells including endothelial progenitors undergo extensive migration and differentiation events; however  whether and how these processes are interrelated remains unclear.  The transcription factor Npas4l is necessary for endothelial specification in zebrafish by inducing the expression of the transcription factor genes etsrp  tal1 and lmo2.  We generated a knock in reporter in the npas4l locus to visualize endothelial progenitors and their derivatives in wild type and mutant embryos.  We find that in npas4l mutants  npas4l reporter expressing cells do not migrate to the midline and instead contribute to skeletal muscle and pronephric tubules.  Investigating the Npas4l transcriptional effectors  we find that npas4l reporter expressing cells in tal1 mutants fail to migrate while those in etsrp mutants migrate but fail to differentiate.  In lmo2 mutants  npas4l reporter expressing cells migrate and differentiate  but many express pronephric tubule markers.  Altogether  these data reveal the complex regulation of endothelial progenitor migration  differentiation and fate restriction. Overall design: Comparison of npas4l reporter positive cells from heterozygous and homozygous npas4l mutant embryos using single cell RNAseq on sorted cells.,,,,npas4l mut,GSM5070576,,tissue:npas4l reporter positive cells|cell type:sorted npas4l reporter positive cells|Stage:20 hpf,npas4l mut,Reads were aligned against the zebrafish genome DanRer11 and counted by StarSolo  followed by secondary analysis in Annotated Data Format. Preprocessed counts were further analysed using Scanpy. Basic cell quality control was conducted by taking the number of detected genes and mitochondrial content into consideration. We removed only 16 cells in total that did not express between 1000 and 7000 genes or had an mitochondrial content less than 6%. Further  we filtered genes if they were detected in less than 30 cells <0.3%. Raw counts per cell were normalised to the median count over all cells and transformed into log space to stabilise variance. We initially reduced dimensionality of the dataset using PCA  retaining 50 principal components. Subsequent steps  like low dimensional UMAP embedding and cell clustering via community detection  were based on the initial PCA. Final data visualization was done by scVelo and cellxgene packages. Genome build: DanRer11 Supplementary files format and content: Mattonet adata obs.csv: basic anndata observations Sample  n genes by counts  log1p total counts  pct counts is mito  n genes detected  leiden clustering Supplementary files format and content: Mattonet udata obs.csv: velocity anmdata observations initial size unspliced  initial size spliced  initial size  n counts  velocity self transition  root cells  end points  velocity pseudotime,npas4l reporter positive cells,,Tails from 150 npas4l heterozygous npas4lbns423/+ and trans heterozygous npas4lbns423/bns297 mutant embryos were cut at the anterior end of the yolk extension at 20 hpf in DMEM/F10+5%FBS+0.01% tricaine on agarose coated plates. The cells were dissociated using the Pierce Cardiomyocyte dissociation kit according to manufacturer instructions and FACS sorted for GFP fluorescence using an AriaIII sorter and DAPI as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer’s protocol to obtain 5000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10xGenomics. Single cell RNAseq library preparation was done using standard protocol.,,cell type:sorted npas4l reporter positive cells|Stage:20 hpf,GSM5070576,GSM5070576: npas4l mut; Danio rerio; RNA Seq,GSM5070576,,1,Tails from 150 npas4l heterozygous npas4lbns423/+ and trans heterozygous npas4lbns423/bns297 mutant embryos were cut at the anterior end of the yolk extension at 20 hpf in DMEM/F10+5%FBS+0.01% tricaine on agarose coated plates. The cells were dissociated using the Pierce Cardiomyocyte dissociation kit according to manufacturer instructions and FACS sorted for GFP fluorescence using an AriaIII sorter and DAPI as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer's protocol to obtain 5000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10xGenomics. Single cell RNAseq library preparation was done using standard protocol.,GEO Accession:GSM5070576,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP305389,,,Kenny_scRNA_Mut_BC.fastq.gz Kenny_scRNA_Mut_cDNA.fastq.gz,fastq fastq,15859812024.0,188807286.0,GSM5070576 r1,0:28 1:56,A:4422689365;C:3447909764;G:3545664942;T:4438912669;N:4635284,28,56,,,4422689365,3447909764,3545664942,4438912669,4635284,SRX10052201,SRS8215327,SRA1193720,GEO,MPI for heart and lung research,2,0.00208,0.92256,0.00079,0.14522,0.99602,0.81554,0.38888,0.49915,28,56,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2021-02-08,Segmentation,Embryo,Multi-tissue,Multi-system
63227,SRR13660515,SRX10052200,SRS8215328,SRP305389,PRJNA700672,Endothelial progenitor migration and differentiation is regulated by distinct transcriptional effectors of Cloche/Npas4l,GSE166396,Transcriptome Analysis,Embryonic cells including endothelial progenitors undergo extensive migration and differentiation events; however  whether and how these processes are interrelated remains unclear.  The transcription factor Npas4l is necessary for endothelial specification in zebrafish by inducing the expression of the transcription factor genes etsrp  tal1 and lmo2.  We generated a knock in reporter in the npas4l locus to visualize endothelial progenitors and their derivatives in wild type and mutant embryos.  We find that in npas4l mutants  npas4l reporter expressing cells do not migrate to the midline and instead contribute to skeletal muscle and pronephric tubules.  Investigating the Npas4l transcriptional effectors  we find that npas4l reporter expressing cells in tal1 mutants fail to migrate while those in etsrp mutants migrate but fail to differentiate.  In lmo2 mutants  npas4l reporter expressing cells migrate and differentiate  but many express pronephric tubule markers.  Altogether  these data reveal the complex regulation of endothelial progenitor migration  differentiation and fate restriction. Overall design: Comparison of npas4l reporter positive cells from heterozygous and homozygous npas4l mutant embryos using single cell RNAseq on sorted cells.,,,,npas4l het,GSM5070575,,tissue:npas4l reporter positive cells|cell type:sorted npas4l reporter positive cells|Stage:20 hpf,npas4l het,Reads were aligned against the zebrafish genome DanRer11 and counted by StarSolo  followed by secondary analysis in Annotated Data Format. Preprocessed counts were further analysed using Scanpy. Basic cell quality control was conducted by taking the number of detected genes and mitochondrial content into consideration. We removed only 16 cells in total that did not express between 1000 and 7000 genes or had an mitochondrial content less than 6%. Further  we filtered genes if they were detected in less than 30 cells <0.3%. Raw counts per cell were normalised to the median count over all cells and transformed into log space to stabilise variance. We initially reduced dimensionality of the dataset using PCA  retaining 50 principal components. Subsequent steps  like low dimensional UMAP embedding and cell clustering via community detection  were based on the initial PCA. Final data visualization was done by scVelo and cellxgene packages. Genome build: DanRer11 Supplementary files format and content: Mattonet adata obs.csv: basic anndata observations Sample  n genes by counts  log1p total counts  pct counts is mito  n genes detected  leiden clustering Supplementary files format and content: Mattonet udata obs.csv: velocity anmdata observations initial size unspliced  initial size spliced  initial size  n counts  velocity self transition  root cells  end points  velocity pseudotime,npas4l reporter positive cells,,Tails from 150 npas4l heterozygous npas4lbns423/+ and trans heterozygous npas4lbns423/bns297 mutant embryos were cut at the anterior end of the yolk extension at 20 hpf in DMEM/F10+5%FBS+0.01% tricaine on agarose coated plates. The cells were dissociated using the Pierce Cardiomyocyte dissociation kit according to manufacturer instructions and FACS sorted for GFP fluorescence using an AriaIII sorter and DAPI as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer’s protocol to obtain 5000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10xGenomics. Single cell RNAseq library preparation was done using standard protocol.,,cell type:sorted npas4l reporter positive cells|Stage:20 hpf,GSM5070575,GSM5070575: npas4l het; Danio rerio; RNA Seq,GSM5070575,,1,Tails from 150 npas4l heterozygous npas4lbns423/+ and trans heterozygous npas4lbns423/bns297 mutant embryos were cut at the anterior end of the yolk extension at 20 hpf in DMEM/F10+5%FBS+0.01% tricaine on agarose coated plates. The cells were dissociated using the Pierce Cardiomyocyte dissociation kit according to manufacturer instructions and FACS sorted for GFP fluorescence using an AriaIII sorter and DAPI as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer's protocol to obtain 5000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 10xGenomics. Single cell RNAseq library preparation was done using standard protocol.,GEO Accession:GSM5070575,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP305389,,,Kenny_scRNA_Het_BC.fastq.gz Kenny_scRNA_Het_cDNA.fastq.gz,fastq fastq,21384880020.0,254581905.0,GSM5070575 r1,0:28 1:56,A:5828925615;C:4816199698;G:4885826464;T:5847660976;N:6267267,28,56,,,5828925615,4816199698,4885826464,5847660976,6267267,SRX10052200,SRS8215328,SRA1193720,GEO,MPI for heart and lung research,2,0.00213,0.9288,0.00071,0.11864,0.99559,0.8312,0.43162,0.47914,28,56,T,B,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Germany,2021-02-08,Segmentation,Embryo,Multi-tissue,Multi-system
63253,SRR13693851,SRX10083149,SRS8241165,SRP305927,PRJNA701473,Single cell RNA seq analysis for normal lymphocytes and IRF4 driven zebrafish tumors,GSE166646,Transcriptome Analysis,Single cell RNA seq analysis was carried out for 1 normal sample from a control zebrafish line lck mCherry and 5 non tumor and 14 tumor samples from the IRF4 transgenic line lck IRF4.  Tumor cells were harvested from the animals with or without xxx p53 mutation at different time points of tumor development. Overall design: Human IRF4 gene was overexpressed together with mCherry under the zebrafish lck promoter.  Samples were harvested from control or IRF4 transgenic zebrafish lck IRF4 with or without xxx p53 mutation. mCherry positive cells sorted by flow cytometry.  Normal cells was prepared from a control zebrafish which overexpresses only mCherry.  Single cell RNA seq analysis was performed by 10X Genomics using three prime Chromium Single Cell Technique.,parent bioproject:PRJNA701471,pubmed:35504924,,Normal cells  Control scRNA Seq,GSM5076939,,source name:Zebrafish cells  control|tissue:normal cells|sorted cells:mCherry sorted cells|genotype:Control,Normal cells  Control scRNA Seq,Annotations from Ensembl GRCz11 for zebrafish genes and transcripts were used and concatenated with mCherry and human IRF4 ENSG00000137265 gene information.  Single cell data were generated using mkref from cellranger package. Alignment  barcode assignation  and generation of cell gene count matrices were all performed using cellranger count from 10x genomics version 3.1.0 cellranger count   id zv11 {sampleID}   fastqs={directory scSeq}   sample={sampleID}  transcriptome={mkgtf generated STAR library} Genome build: GRCz11 Danio Rerio + mCherry + Human IRF4 ENSG00000137265 Supplementary files format and content: HDF5 Gene Barcode Matrix format containing UMI counts of each gene for individual cells.  The data files can be deconvoluted to tsv and mtx files using cellranger reanalyze,Zebrafish cells  control,No treatment,post sorting  the cells were washed and processed for barcoding using the 10x Genomics Chromium Single Cell three prime Solution v2 kit following manufacturer's instructions. Illumina P5 and P7 sequences and sample index sequences are added during the Sample Index PCR. The final library fragments contain the P5  P7  Read 1 and Read 2 sequences used in Illumina bridge amplification and sequencing. Additionally  each fragment contains the 10x Barcode  UMI and cDNA insert sequence used in data analysis. Single cell RNA Seq,The human IRF4 gene and mCherry fluorescent marker gene were transduced under the zebrafish lck promoter in zebrafish.  post the development of lymphoma phenotype  tumor cells were harvested and sorted by flow cytometry based on mCherry fluorescence expression.  A zebrafish which was transduced only with mCherry was used as a control.,tissue:normal cells|sorted cells:mCherry sorted cells|genotype:Control,GSM5076939,GSM5076939: Normal cells  Control scRNA Seq; Danio rerio; RNA Seq,GSM5076939,,1,post sorting  the cells were washed and processed for barcoding using the 10x Genomics Chromium Single Cell three prime Solution v2 kit following manufacturer's instructions. Illumina P5 and P7 sequences and sample index sequences are added during the Sample Index PCR. The final library fragments contain the P5  P7  Read 1 and Read 2 sequences used in Illumina bridge amplification and sequencing. Additionally  each fragment contains the 10x Barcode  UMI and cDNA insert sequence used in data analysis. Single cell RNA Seq,GEO Accession:GSM5076939,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,HiSeq X Ten,,SRP305927,,loader:fastq load.py|options:  platform=Illumina   readTypes=TTB   read1PairFiles=PC8 I1.fastq.gz   read2PairFiles=PC8 R1.fastq.gz   read3PairFiles=PC8 R2.fastq.gz,PC8_I1.fastq.gz PC8_R1.fastq.gz PC8_R2.fastq.gz,fastq fastq fastq,27368502216.0,207337138.0,GSM5076939 r1,0:8 1:26 2:98,A:7549832339;C:6248269949;G:6287147333;T:7274505713;N:8746882,8,26,98,,7549832339,6248269949,6287147333,7274505713,8746882,SRX10083149,SRS8241165,SRA1195246,GEO,"Takaomi Sanda Lab, Cancer Science Institute, National University of Singapore",1,0.89463,,0.1714,,0.81093,,0.53939,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,Singapore,2021-02-11,Undetermined,Undetermined,Multi-tissue,Multi-system
66718,SRR16490737,SRX12693840,SRS10644723,SRP342163,PRJNA772751,Single cell transcriptomic atlas reveals the evolution of immune cells and molecules across multiple vertebrate species,GSE186158,Other,We utilized single cell RNA sequencing scRNA seq to carry out comparative transcriptome analysis in various immune cells from the spleens across seven vertebrate species. Overall design: High dimensional single cell RNA sequencing 10X Genomics was used to profile the transcriptomes of total immune cells isolated from seven organism  spleens.,,pubmed:36871615,,Fish,GSM5639492,,tissue:Fish splenocytes|cell type:Splenocytes,Fish,The 10x Genomics Cell Ranger 3.0.1 version pipeline was used to demultiplex raw files into FASTQ files  extract barcodes and UMI  filter  and map reads to the corresponding reference genome  and generate a matrix containing UMI counts. A 10x Genomics Cell Ranger pipeline was used to identify clonotypes by alignment and annotation with the default settings. Genome build: Fish:GRCz11  Frog:Xenopus laevis v2  Mouse:GRCm38  Rat:Rnor 6.0  Pig:Sscrofa11  Monkey:Mmul 8  Human:GRCh38 Supplementary files format and content: Supplementary files format and content: filtered feature bc matrix.tar contains counts matrix stored in the Market Exchange Format for sparse matrices.,Fish splenocytes,Splenocytes were sorted from the spleens of  seven vertebrate species,Spleen tissues were processed with the flat end of a syringe in a 100 mm culture dish containing 5 ml cold FACS buffer 2% FBS in PBS  then passed through a 70 μm cell strainer into a 15 ml tube. Cells were centrifuged to remove the supernatant. Cell pellets were treated with 1ml ACK Ammonium Chloride Potassium Lysing Buffer to remove the red blood cells. post washing with 10 ml cold FACS buffer  the remaining cells were stained with 7AAD Part 76332; Lot B226294 Biolegend for 30 min at 4 °C  then washed and resuspended in cold FACS buffer for flow cytometric sorting using FACS Aria II Cell Sorter BD Biosciences. Sorted 7AAD  cells with a viability higher than 90% were used for 10X genomics scRNA seq. Finally  libraries were sequenced on a NovaSeq 6000 system with NovaSeq 6000 S4 Reagent Kit 300 cycles. RNA libraries were prepared for sequencing using standard Illumina protocols,,cell type:Splenocytes,GSM5639492,GSM5639492: Fish; Danio rerio; RNA Seq,GSM5639492 r1,GSM5639492,1,Spleen tissues were processed with the flat end of a syringe in a 100 mm culture dish containing 5 ml cold FACS buffer 2% FBS in PBS  then passed through a 70 μm cell strainer into a 15 ml tube. Cells were centrifuged to remove the supernatant. Cell pellets were treated with 1ml ACK Ammonium Chloride Potassium Lysing Buffer to remove the red blood cells. post washing with 10 ml cold FACS buffer  the remaining cells were stained with 7AAD Part 76332; Lot B226294 Biolegend for 30 min at 4 °C  then washed and resuspended in cold FACS buffer for flow cytometric sorting using FACS Aria II Cell Sorter BD Biosciences. Sorted 7AAD  cells with a viability higher than 90% were used for 10X genomics scRNA seq. Finally  libraries were sequenced on a NovaSeq 6000 system with NovaSeq 6000 S4 Reagent Kit 300 cycles. RNA libraries were prepared for sequencing using standard Illumina protocols,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP342163,,,fish5_S1_L002_R1_001.fastq.gz fish5_S1_L002_R2_001.fastq.gz,fastq fastq,128052138306.0,424013703.0,GSM5639492 r1,0:151 1:151,A:31857061588;C:30793041207;G:30850699878;T:34545754656;N:5580977,151,151,,,31857061588,30793041207,30850699878,34545754656,5580977,SRX12693840,SRS10644723,SRA1377133,xian jiaotong university,xian jiaotong university,2,0.06618,0.94142,0.01504,0.09555,0.96631,0.81266,0.69482,0.58057,151,151,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2021-10-19,Undetermined,Undetermined,Multi-tissue,Multi-system
67596,SRR17218109,SRX13397704,SRS11300866,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  5,GSM5732076,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  5,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732076,GSM5732076: KM  Myeloid  5; Danio rerio; RNA Seq,GSM5732076,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732076,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_27_myeloid_S4_L001_I1_001.fastq.gz fish_27_myeloid_S4_L001_R1_001.fastq.gz fish_27_myeloid_S4_L001_R2_001.fastq.gz,fastq fastq fastq,11749364895.0,93156582.0,GSM5732076 r1,0:8 1:27.59 2:90.54,A:2391406507;C:1812685040;G:1971247879;T:2258743152;N:195740,8,27,90,,2391406507,1812685040,1971247879,2258743152,195740,SRX13397704,SRS11300866,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90857,,0.08472,,0.85518,,0.48289,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67597,SRR17218110,SRX13397704,SRS11300866,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  5,GSM5732076,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  5,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732076,GSM5732076: KM  Myeloid  5; Danio rerio; RNA Seq,GSM5732076,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732076,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_27_myeloid_S4_L002_I1_001.fastq.gz fish_27_myeloid_S4_L002_R1_001.fastq.gz fish_27_myeloid_S4_L002_R2_001.fastq.gz,fastq fastq fastq,12092224776.0,95875357.0,GSM5732076 r2,0:8 1:27.59 2:90.54,A:2455357360;C:1869720709;G:2037119856;T:2318012173;N:200034,8,27,90,,2455357360,1869720709,2037119856,2318012173,200034,SRX13397704,SRS11300866,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90899,,0.0844,,0.85596,,0.44313,,90,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67598,SRR17218107,SRX13397703,SRS11300864,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  5,GSM5732075,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  5,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732075,GSM5732075: KM  Lym  Prog  5; Danio rerio; RNA Seq,GSM5732075,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732075,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_27_lymphoid_S3_L001_I1_001.fastq.gz fish_27_lymphoid_S3_L001_R1_001.fastq.gz fish_27_lymphoid_S3_L001_R2_001.fastq.gz,fastq fastq fastq,13496808543.0,107030769.0,GSM5732075 r1,0:8 1:27.58 2:90.52,A:2626946378;C:2274484102;G:2349900648;T:2436769052;N:217231,8,27,90,,2626946378,2274484102,2349900648,2436769052,217231,SRX13397703,SRS11300864,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.91164,,0.07594,,0.8448,,0.55371,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67599,SRR17218108,SRX13397703,SRS11300864,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  5,GSM5732075,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  5,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4.5 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732075,GSM5732075: KM  Lym  Prog  5; Danio rerio; RNA Seq,GSM5732075,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732075,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_27_lymphoid_S3_L002_I1_001.fastq.gz fish_27_lymphoid_S3_L002_R1_001.fastq.gz fish_27_lymphoid_S3_L002_R2_001.fastq.gz,fastq fastq fastq,13953104861.0,110649272.0,GSM5732075 r2,0:8 1:27.58 2:90.52,A:2711848837;C:2355083508;G:2437754950;T:2510947800;N:231356,8,27,90,,2711848837,2355083508,2437754950,2510947800,231356,SRX13397703,SRS11300864,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90963,,0.07451,,0.84555,,0.45045,,90,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67600,SRR17218105,SRX13397702,SRS11300863,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  4,GSM5732074,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  4,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732074,GSM5732074: KM  Myeloid  4; Danio rerio; RNA Seq,GSM5732074,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732074,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_49_myeloid_S4_L001_I1_001.fastq.gz fish_49_myeloid_S4_L001_R1_001.fastq.gz fish_49_myeloid_S4_L001_R2_001.fastq.gz,fastq fastq fastq,10086144160.0,79960137.0,GSM5732074 r1,0:8 1:27.59 2:90.55,A:2076273328;C:1559926460;G:1700451001;T:1903120126;N:458340,8,27,90,,2076273328,1559926460,1700451001,1903120126,458340,SRX13397702,SRS11300863,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92732,,0.09687,,0.85587,,0.52284,,89,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67601,SRR17218106,SRX13397702,SRS11300863,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  4,GSM5732074,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  4,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732074,GSM5732074: KM  Myeloid  4; Danio rerio; RNA Seq,GSM5732074,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732074,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_49_myeloid_S4_L002_I1_001.fastq.gz fish_49_myeloid_S4_L002_R1_001.fastq.gz fish_49_myeloid_S4_L002_R2_001.fastq.gz,fastq fastq fastq,10078550888.0,79899896.0,GSM5732074 r2,0:8 1:27.59 2:90.55,A:2074834444;C:1558864172;G:1698990089;T:1901697415;N:388878,8,27,90,,2074834444,1558864172,1698990089,1901697415,388878,SRX13397702,SRS11300863,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92761,,0.09685,,0.85543,,0.52231,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67602,SRR17218103,SRX13397701,SRS11300861,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  4,GSM5732073,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  4,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732073,GSM5732073: KM  Lym  Prog  4; Danio rerio; RNA Seq,GSM5732073,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732073,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_49_lymphoid_S3_L001_I1_001.fastq.gz fish_49_lymphoid_S3_L001_R1_001.fastq.gz fish_49_lymphoid_S3_L001_R2_001.fastq.gz,fastq fastq fastq,23667853878.0,187695182.0,GSM5732073 r1,0:8 1:27.59 2:90.51,A:4561315827;C:4042818801;G:4102327804;T:4280838818;N:1075348,8,27,90,,4561315827,4042818801,4102327804,4280838818,1075348,SRX13397701,SRS11300861,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90678,,0.07736,,0.84711,,0.45832,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67603,SRR17218104,SRX13397701,SRS11300861,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  4,GSM5732073,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  4,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732073,GSM5732073: KM  Lym  Prog  4; Danio rerio; RNA Seq,GSM5732073,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732073,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_49_lymphoid_S3_L002_I1_001.fastq.gz fish_49_lymphoid_S3_L002_R1_001.fastq.gz fish_49_lymphoid_S3_L002_R2_001.fastq.gz,fastq fastq fastq,23656514553.0,187605835.0,GSM5732073 r2,0:8 1:27.59 2:90.51,A:4559082739;C:4041456222;G:4099953705;T:4278831382;N:913359,8,27,90,,4559082739,4041456222,4099953705,4278831382,913359,SRX13397701,SRS11300861,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90701,,0.07716,,0.84904,,0.46314,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67604,SRR17218101,SRX13397700,SRS11300862,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  3,GSM5732072,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  3,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732072,GSM5732072: KM  Myeloid  3; Danio rerio; RNA Seq,GSM5732072,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732072,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_44_myeloid_S2_L001_I1_001.fastq.gz fish_44_myeloid_S2_L001_R1_001.fastq.gz fish_44_myeloid_S2_L001_R2_001.fastq.gz,fastq fastq fastq,14003996837.0,110994802.0,GSM5732072 r1,0:8 1:27.59 2:90.58,A:2979488967;C:2068071790;G:2214121174;T:2791446091;N:636953,8,27,90,,2979488967,2068071790,2214121174,2791446091,636953,SRX13397700,SRS11300862,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90083,,0.12109,,0.84542,,0.52606,,89,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67605,SRR17218102,SRX13397700,SRS11300862,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  3,GSM5732072,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  3,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732072,GSM5732072: KM  Myeloid  3; Danio rerio; RNA Seq,GSM5732072,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732072,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_44_myeloid_S2_L002_I1_001.fastq.gz fish_44_myeloid_S2_L002_R1_001.fastq.gz fish_44_myeloid_S2_L002_R2_001.fastq.gz,fastq fastq fastq,13994707212.0,110921280.0,GSM5732072 r2,0:8 1:27.59 2:90.58,A:2977327088;C:2067049220;G:2212642231;T:2789521107;N:543309,8,27,90,,2977327088,2067049220,2212642231,2789521107,543309,SRX13397700,SRS11300862,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.90039,,0.12104,,0.84524,,0.52306,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67606,SRR17218099,SRX13397699,SRS11300860,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  3,GSM5732071,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  3,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732071,GSM5732071: KM  Lym  Prog  3; Danio rerio; RNA Seq,GSM5732071,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732071,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_44_lymphoid_S1_L001_I1_001.fastq.gz fish_44_lymphoid_S1_L001_R1_001.fastq.gz fish_44_lymphoid_S1_L001_R2_001.fastq.gz,fastq fastq fastq,13094416737.0,103839618.0,GSM5732071 r1,0:8 1:27.59 2:90.52,A:2531961623;C:2230900332;G:2254536942;T:2381097817;N:599436,8,27,90,,2531961623,2230900332,2254536942,2381097817,599436,SRX13397699,SRS11300860,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.88993,,0.07116,,0.85567,,0.47698,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67607,SRR17218100,SRX13397699,SRS11300860,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  3,GSM5732071,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  3,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732071,GSM5732071: KM  Lym  Prog  3; Danio rerio; RNA Seq,GSM5732071,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732071,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,fish_44_lymphoid_S1_L002_I1_001.fastq.gz fish_44_lymphoid_S1_L002_R1_001.fastq.gz fish_44_lymphoid_S1_L002_R2_001.fastq.gz,fastq fastq fastq,13086686536.0,103778772.0,GSM5732071 r2,0:8 1:27.59 2:90.52,A:2530342591;C:2229880925;G:2253019984;T:2379793583;N:502900,8,27,90,,2530342591,2229880925,2253019984,2379793583,502900,SRX13397699,SRS11300860,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.88991,,0.07094,,0.85504,,0.47263,,90,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67610,SRR17218095,SRX13397697,SRS11300858,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  2,GSM5732069,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  2,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732069,GSM5732069: KM  Myeloid  2; Danio rerio; RNA Seq,GSM5732069,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732069,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_24_Myeloid__S2_L001_I1_001.fastq.gz Fish_24_Myeloid__S2_L001_R1_001.fastq.gz Fish_24_Myeloid__S2_L001_R2_001.fastq.gz,fastq fastq fastq,12723767840.0,100866379.0,GSM5732069 r1,0:8 1:27.59 2:90.56,A:2661308081;C:1888190203;G:2104404770;T:2480108455;N:231064,8,27,90,,2661308081,1888190203,2104404770,2480108455,231064,SRX13397697,SRS11300858,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.93294,,0.09284,,0.85054,,0.48913,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67611,SRR17218096,SRX13397697,SRS11300858,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  2,GSM5732069,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  2,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732069,GSM5732069: KM  Myeloid  2; Danio rerio; RNA Seq,GSM5732069,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732069,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_24_Myeloid__S2_L002_I1_001.fastq.gz Fish_24_Myeloid__S2_L002_R1_001.fastq.gz Fish_24_Myeloid__S2_L002_R2_001.fastq.gz,fastq fastq fastq,12676361798.0,100490505.0,GSM5732069 r2,0:8 1:27.59 2:90.56,A:2652256616;C:1880288783;G:2095102149;T:2472303748;N:259945,8,27,90,,2652256616,1880288783,2095102149,2472303748,259945,SRX13397697,SRS11300858,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.93404,,0.09191,,0.85036,,0.48617,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67612,SRR17218093,SRX13397696,SRS11300856,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  2,GSM5732068,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  2,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732068,GSM5732068: KM  Lym  Prog  2; Danio rerio; RNA Seq,GSM5732068,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732068,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_24_Lymphoid__S1_L001_I1_001.fastq.gz Fish_24_Lymphoid__S1_L001_R1_001.fastq.gz Fish_24_Lymphoid__S1_L001_R2_001.fastq.gz,fastq fastq fastq,15733686154.0,124760148.0,GSM5732068 r1,0:8 1:27.58 2:90.53,A:3104338688;C:2600154759;G:2737304416;T:2852392277;N:287465,8,27,90,,3104338688,2600154759,2737304416,2852392277,287465,SRX13397696,SRS11300856,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.95054,,0.09234,,0.8352,,0.51282,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67613,SRR17218094,SRX13397696,SRS11300856,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  2,GSM5732068,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  2,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732068,GSM5732068: KM  Lym  Prog  2; Danio rerio; RNA Seq,GSM5732068,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732068,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_24_Lymphoid__S1_L002_I1_001.fastq.gz Fish_24_Lymphoid__S1_L002_R1_001.fastq.gz Fish_24_Lymphoid__S1_L002_R2_001.fastq.gz,fastq fastq fastq,15649297953.0,124090955.0,GSM5732068 r2,0:8 1:27.58 2:90.53,A:3087971774;C:2585586481;G:2720843685;T:2839166000;N:322202,8,27,90,,3087971774,2585586481,2720843685,2839166000,322202,SRX13397696,SRS11300856,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.94972,,0.09246,,0.83544,,0.52156,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67616,SRR17218089,SRX13397694,SRS11300855,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  1,GSM5732066,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  1,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732066,GSM5732066: KM  Myeloid  1; Danio rerio; RNA Seq,GSM5732066,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732066,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_22_Myeloid__S2_L001_I1_001.fastq.gz Fish_22_Myeloid__S2_L001_R1_001.fastq.gz Fish_22_Myeloid__S2_L001_R2_001.fastq.gz,fastq fastq fastq,13324252515.0,105625411.0,GSM5732066 r1,0:8 1:27.59 2:90.56,A:2768613739;C:2001578304;G:2166922138;T:2627481682;N:616468,8,27,90,,2768613739,2001578304,2166922138,2627481682,616468,SRX13397694,SRS11300855,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92619,,0.09612,,0.84824,,0.51407,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67617,SRR17218090,SRX13397694,SRS11300855,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Myeloid  1,GSM5732066,,tissue:sorted kidney marrow granulocytes|cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Myeloid  1,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow granulocytes,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow granulocytes|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732066,GSM5732066: KM  Myeloid  1; Danio rerio; RNA Seq,GSM5732066,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732066,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_22_Myeloid__S2_L002_I1_001.fastq.gz Fish_22_Myeloid__S2_L002_R1_001.fastq.gz Fish_22_Myeloid__S2_L002_R2_001.fastq.gz,fastq fastq fastq,13390698340.0,106151896.0,GSM5732066 r2,0:8 1:27.59 2:90.56,A:2785609814;C:2010269896;G:2172154902;T:2644451064;N:418590,8,27,90,,2785609814,2010269896,2172154902,2644451064,418590,SRX13397694,SRS11300855,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92458,,0.09669,,0.84745,,0.52988,,90,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67618,SRR17218087,SRX13397693,SRS11300854,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  1,GSM5732065,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  1,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732065,GSM5732065: KM  Lym  Prog  1; Danio rerio; RNA Seq,GSM5732065,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732065,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_22_Lymphoid__S1_L001_I1_001.fastq.gz Fish_22_Lymphoid__S1_L001_R1_001.fastq.gz Fish_22_Lymphoid__S1_L001_R2_001.fastq.gz,fastq fastq fastq,14336274930.0,113694762.0,GSM5732065 r1,0:8 1:27.58 2:90.51,A:2779833433;C:2445043827;G:2529609458;T:2535735722;N:664397,8,27,90,,2779833433,2445043827,2529609458,2535735722,664397,SRX13397693,SRS11300854,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92148,,0.08283,,0.84228,,0.52455,,91,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system
67619,SRR17218088,SRX13397693,SRS11300854,SRP350544,PRJNA788489,Single Cell Analyses Reveal Early Thymic Progenitors and Pre B Cells in the Zebrafish,GSE190794,Transcriptome Analysis,The zebrafish has proven to be a valuable model organism for studying hematopoiesis  but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single cell RNA sequencing on 70 000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus  elucidated B and T cell developmental trajectories  and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic like cell populations and provided evidence in support of the existence of a pre B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies. Overall design: To analyze the composition of the zebrafish primary hematopoietic organs and to characterize immune cell subpopulations  a total of 7 adult zebrafish 3 mpf 5 mpf and 21 juvenile zebrafish 4 wpf were studied. More specifically  kidney marrows only were dissected from 3 adult GESTALT zebrafish  paired kidney marrows and thymi were dissected from 2 adult GESTALT zebrafish  and thymi only were dissected from 2 adult Tglck:eGFP zebrafish. Thymi only were dissected and pooled from 21 juvenile Tglck:eGFP zebrafish. No other pooling as performed. All tissues were subjected to mechanical dissociation  filtering  and cell sorting on a BD FACSAriaII. Live cells from whole thymi and two fractions of live cells from whole kidney marrow sorted based on FSC and SSC characteristics lymphoid and progenitor fraction; granulocyte fraction were collected in preparation for 10x single cell RNA sequencing. A single 10x lane was used for each adult sample  whereas 4 lanes were used for the juvenile thymi to obtain 4 technical replicates.,,pubmed:35938989,,KM  Lym  Prog  1,GSM5732065,,tissue:sorted kidney marrow lymphoid and progenitor cells|cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,KM  Lym  Prog  1,The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline version 3.1.0. The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Genome build: Zebrafish Ensembl GRCz11 reference Supplementary files format and content: h5 count matrices from the NovaSeq platforms,sorted kidney marrow lymphoid and progenitor cells,,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell 3’ v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,Zebrafish were maintained in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee protocols and in line with Animal Resources at Children’s Hospital ARCH guidelines.,cell type:sorted kidney marrow lymphoid and progenitor cells|strain:GESTALT|developmental stage:Adult|age:4 mpf bleed:yes|dissection buffer:0.5% BSA in DPBS  no Ca2+ or Mg2+|dissection temperature:on ice,GSM5732065,GSM5732065: KM  Lym  Prog  1; Danio rerio; RNA Seq,GSM5732065,,1,Adult and juvenile zebrafish were euthanized by rapid chilling and confirmed deceased prior to dissecting thymi and/or kidney marrows. Live cells were sorted 85 um nozzle on a BD FACSAriaII into 300 μL of 0.5% BSA in DPBS and kept on ice following the sort. Between 50 000 and 100 000 cells were sorted per sample  assessed by Trypan blue staining  and then pelleted by centrifugation at 400g x 5 minutes and resuspended to a final concentration of 1000 cells/uL. Transcriptome profiling was performed using the Chromium Next GEM Single Cell three prime v3.1 Reagent Kits 10x Genomics  Inc.. A targeted recovery of 6 000 cells was set for loading the Chromium Controller in all assays and steps were performed as described in the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 User Guide Rev D. The final transcriptome libraries were pooled in sets of 4 or 5 and sequenced at the Harvard University Bauer Core https://bauercore.fas.harvard.edu/ on an Illumina NovaSeq 6000 using an SP flow cell Read 1: 28 cycles  i7 index: 8 cycles  i5 index: 0 cycles  Read 2: 91 cycles. scRNA Seq,GEO Accession:GSM5732065,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP350544,,loader:fastq load.py,Fish_22_Lymphoid__S1_L002_I1_001.fastq.gz Fish_22_Lymphoid__S1_L002_R1_001.fastq.gz Fish_22_Lymphoid__S1_L002_R2_001.fastq.gz,fastq fastq fastq,14224072051.0,112805330.0,GSM5732065 r2,0:8 1:27.58 2:90.51,A:2760149838;C:2425717356;G:2504208856;T:2519823252;N:437824,8,27,90,,2760149838,2425717356,2504208856,2519823252,437824,SRX13397693,SRS11300854,SRA1342645,GEO,"Oncology/Hematology, Boston Children's Hospital",1,0.92195,,0.08309,,0.84224,,0.54072,,89,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2021-12-13,Adult,Adult,Multi-tissue,Multi-system