{"database": "metadata", "table": "run_metadata", "rows": [[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"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["11131"], "units": {}, "query_ms": 14.390249998541549}