run_metadata: 11132

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
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		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 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 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. 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 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 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									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 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 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. 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 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 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		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			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		Sweden	2022-07-22	Multi-stage	Embryo	Whole Organism	All anatomical structures