{"database": "metadata", "table": "run_metadata", "is_view": false, "human_description_en": "where experiment.library_layout = \"PAIRED\", experiment.library_selection = \"size fractionation\" and experiment.library_source = \"TRANSCRIPTOMIC SINGLE CELL\"", "rows": [[10403, "ERR8527374", "ERX8137561", "ERS10539829", "ERP135430", "PRJEB50826", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E-MTAB-11079_2", "Transcriptome Analysis", "Libraries were made to compare the transcriptome of renin lineage cells RLCs from wild type versus ren knockout zebrafish kidneys. RLCs were FAC sorted from pooled kidneys of ren+/+ or ren / zebrafish which carried ren:RFP and acta2:EGFP reporter genes allowing the isolation of renin expressing cells and smooth muscle cells.", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", null, "Protocols: Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "ZF2", "SAMEA12941033", "University Of Edinburgh", "ENA first public:2022 06 10|ENA last update:2022 06 10|External Id:SAMEA12941033|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2022 06 10T00:18:52Z|INSDC last update:2022 06 10T00:18:52Z|INSDC status:public|Submitter Id:E MTAB 11079 2:ZF2|age:6|broker name:ArrayExpress|cell type:renin lineage cell|common name:zebrafish|developmental stage:adult|genotype:ren:RFP; acta2:EGFP; ren / |individual:9 12 kidneys pooled|inferred cell type:recruited RLC|organism part:kidney|phenotype:ren knockout|sample name:E MTAB 11079 2:ZF2|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E MTAB 11079 2:ZF2 p", "ZF2 p", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "Experimental Factor: phenotype:ren knockout", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "NextSeq 500", null, "ERP135430", "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", "ZF2_S2_L002_I1_001.fastq.gz ZF2_S2_L002_R1_001.fastq.gz ZF2_S2_L002_R2_001.fastq.gz", "fastq fastq fastq", 38557081640.0, 125185330.0, "E MTAB 11079 2:ZF2 S2 L002", "0:8 1:150 2:150", "A:13781315856;C:6436030786;G:6578059630;T:10759631611;N:561117", 8, 150, 150, null, 13781315856, 6436030786, 6578059630, 10759631611, 561117, "ERX8137561", "ERS10539829", "ERA9016154", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 2, 0.0, 0.84449, 0.0, 0.09657, 1.0, 0.8242, null, 0.52635, 150, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "size_fractionation", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2022-06-10", "Adult", "Adult", "Multi-tissue", "Multi-system"], [10404, "ERR8527377", "ERX8137561", "ERS10539829", "ERP135430", "PRJEB50826", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E-MTAB-11079_2", "Transcriptome Analysis", "Libraries were made to compare the transcriptome of renin lineage cells RLCs from wild type versus ren knockout zebrafish kidneys. RLCs were FAC sorted from pooled kidneys of ren+/+ or ren / zebrafish which carried ren:RFP and acta2:EGFP reporter genes allowing the isolation of renin expressing cells and smooth muscle cells.", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", null, "Protocols: Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "ZF2", "SAMEA12941033", "University Of Edinburgh", "ENA first public:2022 06 10|ENA last update:2022 06 10|External Id:SAMEA12941033|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2022 06 10T00:18:52Z|INSDC last update:2022 06 10T00:18:52Z|INSDC status:public|Submitter Id:E MTAB 11079 2:ZF2|age:6|broker name:ArrayExpress|cell type:renin lineage cell|common name:zebrafish|developmental stage:adult|genotype:ren:RFP; acta2:EGFP; ren / |individual:9 12 kidneys pooled|inferred cell type:recruited RLC|organism part:kidney|phenotype:ren knockout|sample name:E MTAB 11079 2:ZF2|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E MTAB 11079 2:ZF2 p", "ZF2 p", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "Experimental Factor: phenotype:ren knockout", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "NextSeq 500", null, "ERP135430", "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", "ZF2_S2_L001_I1_001.fastq.gz ZF2_S2_L001_R1_001.fastq.gz ZF2_S2_L001_R2_001.fastq.gz", "fastq fastq fastq", 38872269128.0, 126208666.0, "E MTAB 11079 2:ZF2 S2 L001", "0:8 1:150 2:150", "A:15151865400;C:6355030057;G:6591732598;T:9763441371;N:530374", 8, 150, 150, null, 15151865400, 6355030057, 6591732598, 9763441371, 530374, "ERX8137561", "ERS10539829", "ERA9016154", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 2, 0.0, 0.84526, 0.0, 0.09779, 1.0, 0.82609, null, 0.54388, 150, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "size_fractionation", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2022-06-10", "Adult", "Adult", "Multi-tissue", "Multi-system"], [10405, "ERR8527376", "ERX8137560", "ERS10539828", "ERP135430", "PRJEB50826", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E-MTAB-11079_2", "Transcriptome Analysis", "Libraries were made to compare the transcriptome of renin lineage cells RLCs from wild type versus ren knockout zebrafish kidneys. RLCs were FAC sorted from pooled kidneys of ren+/+ or ren / zebrafish which carried ren:RFP and acta2:EGFP reporter genes allowing the isolation of renin expressing cells and smooth muscle cells.", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", null, "Protocols: Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "ZF1", "SAMEA12941032", "University Of Edinburgh", "ENA first public:2022 06 10|ENA last update:2022 06 10|External Id:SAMEA12941032|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2022 06 10T00:18:52Z|INSDC last update:2022 06 10T00:18:52Z|INSDC status:public|Submitter Id:E MTAB 11079 2:ZF1|age:6|broker name:ArrayExpress|cell type:renin lineage cell|common name:zebrafish|developmental stage:adult|genotype:ren:RFP; acta2:EGFP|individual:9 12 kidneys pooled|inferred cell type:juxtaglomerular cell|organism part:kidney|phenotype:wild type|sample name:E MTAB 11079 2:ZF1|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E MTAB 11079 2:ZF1 p", "ZF1 p", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "Experimental Factor: phenotype:wild type", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "NextSeq 500", null, "ERP135430", "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", "ZF1_S1_L002_I1_001.fastq.gz ZF1_S1_L002_R1_001.fastq.gz ZF1_S1_L002_R2_001.fastq.gz", "fastq fastq fastq", 39415722500.0, 127973125.0, "E MTAB 11079 2:ZF1 S1 L002", "0:8 1:150 2:150", "A:14261012964;C:6339158428;G:6467430919;T:11323758989;N:576200", 8, 150, 150, null, 14261012964, 6339158428, 6467430919, 11323758989, 576200, "ERX8137560", "ERS10539828", "ERA9016154", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 2, 0.0, 0.81065, 0.0, 0.08289, 1.0, 0.82434, null, 0.52903, 150, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "size_fractionation", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2022-06-10", "Adult", "Adult", "Multi-tissue", "Multi-system"], [10406, "ERR8527375", "ERX8137560", "ERS10539828", "ERP135430", "PRJEB50826", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E-MTAB-11079_2", "Transcriptome Analysis", "Libraries were made to compare the transcriptome of renin lineage cells RLCs from wild type versus ren knockout zebrafish kidneys. RLCs were FAC sorted from pooled kidneys of ren+/+ or ren / zebrafish which carried ren:RFP and acta2:EGFP reporter genes allowing the isolation of renin expressing cells and smooth muscle cells.", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", null, "Protocols: Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "ZF1", "SAMEA12941032", "University Of Edinburgh", "ENA first public:2022 06 10|ENA last update:2022 06 10|External Id:SAMEA12941032|INSDC center alias:UOE|INSDC center name:University Of Edinburgh|INSDC first public:2022 06 10T00:18:52Z|INSDC last update:2022 06 10T00:18:52Z|INSDC status:public|Submitter Id:E MTAB 11079 2:ZF1|age:6|broker name:ArrayExpress|cell type:renin lineage cell|common name:zebrafish|developmental stage:adult|genotype:ren:RFP; acta2:EGFP|individual:9 12 kidneys pooled|inferred cell type:juxtaglomerular cell|organism part:kidney|phenotype:wild type|sample name:E MTAB 11079 2:ZF1|sex:mixed|strain:WIK", null, null, null, null, null, null, null, null, "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "E MTAB 11079 2:ZF1 p", "ZF1 p", "scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "Mesonephric kidneys were isolated into ice cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease 20mg/ml DNAse 400units/ml Liberase 0.085mg/ml collagenase 2mg/ml and CaCl2 5mM at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence in order to sort renin expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised tricaine methanesulphonate; MS 222 and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly an sgRNA specifically targeting exon 2 of the renin gene was complexed with tracr RNA and Cas9 protein and injected into a one cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren / knockout fish. Nucleic acid was extracted using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell three prime Library and Gel Bead kit v2 10X Genomics PN 120237 and the Chromium Single Cell A Chip kit 10X Genomics PN 120236 according to manufacturers instructions.", "Experimental Factor: phenotype:wild type", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "NextSeq 500", null, "ERP135430", "NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type ZF1 versus CRISPR targeted renin knockout ZF2 zebrafish", "ENA FIRST PUBLIC:2022 06 10|ENA LAST UPDATE:2022 06 10", "ZF1_S1_L001_I1_001.fastq.gz ZF1_S1_L001_R1_001.fastq.gz ZF1_S1_L001_R2_001.fastq.gz", "fastq fastq fastq", 39744721324.0, 129041303.0, "E MTAB 11079 2:ZF1 S1 L001", "0:8 1:150 2:150", "A:15670316830;C:6278039908;G:6505806971;T:10257683184;N:544007", 8, 150, 150, null, 15670316830, 6278039908, 6505806971, 10257683184, 544007, "ERX8137560", "ERS10539828", "ERA9016154", "University Of Edinburgh|European Nucleotide Archive", "University Of Edinburgh|European Nucleotide Archive", 2, 0.0, 0.81031, 0.0, 0.08495, 1.0, 0.82169, null, 0.51724, 150, 150, "T", "B", "mate1 technical by mapping diff", "illumina", "nextseq", "unknown", "size_fractionation", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2022-06-10", "Adult", "Adult", "Multi-tissue", "Multi-system"], [11130, "ERR9979395", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L002_R1_001.fastq.gz P22202_7002_S2_L002_R2_001.fastq.gz", "fastq fastq", 18144406998.0, 153766161.0, "E MTAB 11984:P22202 7002 S2 L002", "0:28 1:90", "A:5277424121;C:3880928155;G:4118706097;T:4866649878;N:698747", 28, 90, null, null, 5277424121, 3880928155, 4118706097, 4866649878, 698747, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.0109, 0.90145, 0.0053, 0.21373, 0.98746, 0.79423, 0.41198, 0.54447, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11131, "ERR9979394", "ERX9520370", "ERS12499848", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 WT GO+BA", "SAMEA110401656", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 2 WT GO+BA p", "Sample 2 WT GO+BA p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7002_S2_L001_R1_001.fastq.gz P22202_7002_S2_L001_R2_001.fastq.gz", "fastq fastq", 18458951574.0, 156431793.0, "E MTAB 11984:P22202 7002 S2 L001", "0:28 1:90", "A:5372920926;C:3946687814;G:4185647697;T:4953205454;N:489683", 28, 90, null, null, 5372920926, 3946687814, 4185647697, 4953205454, 489683, "ERX9520370", "ERS12499848", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01037, 0.90168, 0.0048, 0.21246, 0.98752, 0.79297, 0.42601, 0.54996, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11132, "ERR9979392", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L001_R1_001.fastq.gz P22202_7001_S1_L001_R2_001.fastq.gz", "fastq fastq", 19396994226.0, 164381307.0, "E MTAB 11984:P22202 7001 S1 L001", "0:28 1:90", "A:5665199484;C:4091196594;G:4415211076;T:5224866754;N:520318", 28, 90, null, null, 5665199484, 4091196594, 4415211076, 5224866754, 520318, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01093, 0.89238, 0.00557, 0.232, 0.98764, 0.79444, 0.40806, 0.54781, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11133, "ERR9979393", "ERX9520369", "ERS12499847", "ERP139765", "PRJEB54901", "Single cell RNA sequencing of germ free zebrafish embryos", "E-MTAB-11984", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", null, "Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 WT control", "SAMEA110401655", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "E MTAB 11984:Sample 1 WT control p", "Sample 1 WT control p", "Single cell RNA sequencing of germ free zebrafish embryos", "The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139765", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos", "ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22", "P22202_7001_S1_L002_R1_001.fastq.gz P22202_7001_S1_L002_R2_001.fastq.gz", "fastq fastq", 19063958224.0, 161558968.0, "E MTAB 11984:P22202 7001 S1 L002", "0:28 1:90", "A:5563588848;C:4022930643;G:4344549562;T:5132136743;N:752428", 28, 90, null, null, 5563588848, 4022930643, 4344549562, 5132136743, 752428, "ERX9520369", "ERS12499847", "ERA16483151", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.01118, 0.89308, 0.00573, 0.23128, 0.98737, 0.79354, 0.39264, 0.54552, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-22", "Multi-stage", "Embryo", "Whole Organism", "All anatomical structures"], [11134, "ERR9981089", "ERX9522049", "ERS12503451", "ERP139797", "PRJEB54930", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "E-MTAB-11991", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. The aim is to evaluate the impact of graphene oxide GO on the innate immune system using zebrafish as a model. We previously performed single cell RNA sequencing of germ free zebrafish embryos exposed to GO plus the microbial metabolite butyrate BA. Here we performed a follow up experiment using germ free lck GFP transgenic fish in which the zebrafish were exposed to GO plus BA at xxx dpf. The embryos were then dissociated and subsequently sorted on lck and submitted for single cell RNA sequencing using 10x Genomics.", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", null, "Protocols: The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 lck control", "SAMEA110406305", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23|External Id:SAMEA110406305|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 23T04:33:35Z|INSDC last update:2022 07 23T04:33:35Z|INSDC status:public|Submitter Id:E MTAB 11991:Sample 1 lck control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:larva|disease:normal|genotype:wild type genotype|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11991:Sample 1 lck control|strain:Tglck:GFP", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "E MTAB 11991:Sample 1 lck control p", "Sample 1 lck control p", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139797", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", "P25452_2002_S2_L001_R1_001.fastq.gz P25452_2002_S2_L001_R2_001.fastq.gz", "fastq fastq", 60177283876.0, 509976982.0, "E MTAB 11991:P25452 2002 S2 L001", "0:28 1:90", "A:17254566438;C:13547211711;G:14188233429;T:15185192817;N:2079481", 28, 90, null, null, 17254566438, 13547211711, 14188233429, 15185192817, 2079481, "ERX9522049", "ERS12503451", "ERA16488297", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.00671, 0.93031, 0.00231, 0.11028, 0.9932, 0.82227, 0.30887, 0.58272, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-23", "Larval", "Larval", "Whole Organism", "All anatomical structures"], [11135, "ERR9981090", "ERX9522049", "ERS12503451", "ERP139797", "PRJEB54930", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "E-MTAB-11991", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. The aim is to evaluate the impact of graphene oxide GO on the innate immune system using zebrafish as a model. We previously performed single cell RNA sequencing of germ free zebrafish embryos exposed to GO plus the microbial metabolite butyrate BA. Here we performed a follow up experiment using germ free lck GFP transgenic fish in which the zebrafish were exposed to GO plus BA at xxx dpf. The embryos were then dissociated and subsequently sorted on lck and submitted for single cell RNA sequencing using 10x Genomics.", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", null, "Protocols: The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 1 lck control", "SAMEA110406305", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23|External Id:SAMEA110406305|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 23T04:33:35Z|INSDC last update:2022 07 23T04:33:35Z|INSDC status:public|Submitter Id:E MTAB 11991:Sample 1 lck control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:larva|disease:normal|genotype:wild type genotype|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11991:Sample 1 lck control|strain:Tglck:GFP", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "E MTAB 11991:Sample 1 lck control p", "Sample 1 lck control p", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0", "Experimental Factor: stimulus:n1", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139797", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", "P25452_2002_S2_L002_R1_001.fastq.gz P25452_2002_S2_L002_R2_001.fastq.gz", "fastq fastq", 60597886144.0, 513541408.0, "E MTAB 11991:P25452 2002 S2 L002", "0:28 1:90", "A:17368154358;C:13649089056;G:14296037620;T:15282869484;N:1735626", 28, 90, null, null, 17368154358, 13649089056, 14296037620, 15282869484, 1735626, "ERX9522049", "ERS12503451", "ERA16488297", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.00656, 0.92899, 0.00211, 0.11059, 0.99334, 0.82335, 0.32311, 0.57644, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-23", "Larval", "Larval", "Whole Organism", "All anatomical structures"], [11136, "ERR9981087", "ERX9522048", "ERS12503450", "ERP139797", "PRJEB54930", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "E-MTAB-11991", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. The aim is to evaluate the impact of graphene oxide GO on the innate immune system using zebrafish as a model. We previously performed single cell RNA sequencing of germ free zebrafish embryos exposed to GO plus the microbial metabolite butyrate BA. Here we performed a follow up experiment using germ free lck GFP transgenic fish in which the zebrafish were exposed to GO plus BA at xxx dpf. The embryos were then dissociated and subsequently sorted on lck and submitted for single cell RNA sequencing using 10x Genomics.", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", null, "Protocols: The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 lck GO+BA", "SAMEA110406304", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23|External Id:SAMEA110406304|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 23T04:33:35Z|INSDC last update:2022 07 23T04:33:35Z|INSDC status:public|Submitter Id:E MTAB 11991:Sample 2 lck GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:larva|disease:normal|genotype:wild type genotype|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11991:Sample 2 lck GO+BA|strain:Tglck:GFP", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "E MTAB 11991:Sample 2 lck GO+BA p", "Sample 2 lck GO+BA p", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Experimental Factor: stimulus:graphene oxide 30 microgram per milliliter|Experimental Factor: compound:butyrate|Experimental Factor: dose:2.5", "AMPLICON", "TRANSCRIPTOMIC SINGLE CELL", "size fractionation", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "ERP139797", "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", "P25452_2001_S1_L001_R1_001.fastq.gz P25452_2001_S1_L001_R2_001.fastq.gz", "fastq fastq", 20866120186.0, 176831527.0, "E MTAB 11991:P25452 2001 S1 L001", "0:28 1:90", "A:5933525376;C:4737179804;G:5025711080;T:5168983880;N:720046", 28, 90, null, null, 5933525376, 4737179804, 5025711080, 5168983880, 720046, "ERX9522048", "ERS12503450", "ERA16488297", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", "Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive", 2, 0.00805, 0.92622, 0.00259, 0.11842, 0.99302, 0.83027, 0.2729, 0.57699, 28, 90, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "full_length", "size_fractionation", "trueseq", "sc", "single_cell_droplet", "10x", null, "Sweden", "2022-07-23", "Larval", "Larval", "Whole Organism", "All anatomical structures"], [11137, "ERR9981088", "ERX9522048", "ERS12503450", "ERP139797", "PRJEB54930", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "E-MTAB-11991", "Transcriptome Analysis", "The present study was conducted in the frame of the EU funded Graphene Flagship project. The aim is to evaluate the impact of graphene oxide GO on the innate immune system using zebrafish as a model. We previously performed single cell RNA sequencing of germ free zebrafish embryos exposed to GO plus the microbial metabolite butyrate BA. Here we performed a follow up experiment using germ free lck GFP transgenic fish in which the zebrafish were exposed to GO plus BA at xxx dpf. The embryos were then dissociated and subsequently sorted on lck and submitted for single cell RNA sequencing using 10x Genomics.", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23", null, "Protocols: The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.", "Sample 2 lck GO+BA", "SAMEA110406304", "Institute of Environmental Medicine, Karolinska Institutet", "ENA FIRST PUBLIC:2022 07 23|ENA LAST UPDATE:2022 07 23|External Id:SAMEA110406304|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 23T04:33:35Z|INSDC last update:2022 07 23T04:33:35Z|INSDC status:public|Submitter Id:E MTAB 11991:Sample 2 lck GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:larva|disease:normal|genotype:wild type genotype|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11991:Sample 2 lck GO+BA|strain:Tglck:GFP", null, null, null, null, null, null, null, null, "Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of lck GFP transgenic zebrafish", "E MTAB 11991:Sample 2 lck GO+BA p", "Sample 2 lck GO+BA p", "Single cell RNA sequencing of lck GFP transgenic zebrafish", "The generation of germ free Tglck:GFP zebrafish followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 \u03bcg/mL kanamycin 5 \u03bcg/mL and amphotericin B 250 ng/mL and incubated at 28\u00b0C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28\u00b0C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free Tglck:GFP zebrafish were exposed to the combination of GO 30 \u00b5g/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Fifty larvae were used as one replicate four replicates i.e. two hundred larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 \u03bcL of pre warmed enzyme mix containing 460 \u03bcL of 0.25% trypsin EDTA Gibco and 40 \u03bcL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 \u00b0C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 \u03bcL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 \u03bcm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 \u03bcM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 \u00b0C and gated based on forward scatter and DRAQ7. The DRAQ7 lck+ cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. 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