RNAquarium Metadata Server
Institute Website Terms of Use Privacy Policy

run_metadata: 11136

This data as json

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
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   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 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. 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°C. 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 µg/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 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL 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 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL 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 μm 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 μM 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 °C 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                 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 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. 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°C. 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 µg/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 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL 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 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL 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 μm 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 μM 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 °C 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   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     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   Sweden 2022-07-23 Larval Larval Whole Organism All anatomical structures