run_metadata: 9701

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
9701	ERR3301004	ERX3327071	ERS3389659	ERP115040	PRJEB32363	RNA seq of zebrafish sa12692 mutants against WT controls	E-MTAB-7920	Transcriptome Analysis	Arrhythmogenic Right Ventricular Cardiomyopathy is a congenital heart disorder characterized by fibrofatty replacement of the myocardium. The exact molecular mechanisms underlying the disease remain to be elucidated and treatment options are limited. The sa12692 mutant line contains a splice site mutation in the plakoglobin gene resulting in the expression of a truncated protein. This protein is highly similar to the protein expressed in Naxos disease a recessive form of ARVC. RNA seq was used to investigate the effect of the sa12692 mutation on gene expression in order to uncover signalling pathways involved in the pathogenesis of ARVC. Gene expression was examined in whole larvae at 5 dpf and in hearts of 1 year old adult fish. Larvae at 5 dpf were selected as this timepoint is equivalent to birth in humans. Adult hearts were selected as ARVC is a disorder of the heart and cardiac symptoms generally manifest in maturity. Hence the molecular effect of the mutation could be profiled at two life stages.	ENA FIRST PUBLIC:2021 04 30\|ENA LAST UPDATE:2019 04 30		Protocols: Larvae were euthanized by rapid freezing. Ten larvae were pooled per sample and homogenized in TRIzol®. All centrifugation steps for RNA extractions occurred at 4°C. RNA was extracted using the TRIzol® method. Pools of 10 zebrafish larvae per biological replicate were collected at 5 dpf and placed in a 1.5 mL tube. Two adult hearts were pooled per biological replicate; these were transferred from RNAlater® to a 1.5 mL tube. TRIzol® 250 μL was added to each tube. The samples were homogenized thoroughly using a sterile 21G needle and a 1 mL syringe. Samples were incubated for 5 minutes at room temperature RT. Chloroform was added one fifth of the volume of TRIzol® used followed by immediate agitation for 15 seconds and incubation at RT for 3 minutes. For phase separation samples were centrifuged at 12000 g for 20 min. The upper aqueous phase containing the RNA was removed carefully and transferred to a new tube. Isopropanol was added half the volume of TRIzol® used and mixed gently. Samples were incubated for 10 minutes at RT and centrifuged at 18000 g for 10 minutes. The supernatant was removed and the pellet was washed in 70% ice cold ethanol one tenth of the volume of TRIzol® used followed by centrifugation at 18000 g for 10 minutes. The ethanol was removed and the pellet was allowed to dry at RT for 7 minutes. The pellet was resuspended in 88 µL of nuclease free water and heated at 55°C for 7 minutes to fully resuspend the pellet. To remove any remaining DNA 2 µL 4 units of DNase enzyme and 10 µL of 10X DNase buffer were added and the samples were incubated for 10 minutes at 37°C. Phenol 200 µL and 20 µL of sodium acetate were added and the samples were centrifuged at 18000 g for 5 minutes. The upper phase was transferred to a new tube and 200 µL of chloroform:isoamyl alcohol 24:1 was added followed by centrifugation at 18000 g for 5 minutes. The chloroform:isoamyl alcohol step was repeated. The upper phase was transferred to a new tube and 200 µL of isopropanol was added to precipitate the RNA. The samples were incubated at 80°C for 30 minutes followed by centrifugation at 18000 g for 30 minutes. The isopropanol was discarded and 200 µL of ice cold ethanol was added to wash the pellet. The samples were centrifuged for a final 5 minutes at 18000 g. The ethanol was discarded and the pellets were air dried for 5 10 minutes. The samples were resuspended in 20 µL nuclease free water. The RNA was stored at 80°C. The quality/integrity of the RNA was assessed with the use of an Agilent RNA 6000 nano kit on an Agilent Bioanalyzer 2100 by following the manufacturer's instructions. RNA integrity was measured by a software tool on the Agilent Bioanalyzer which calculates an RNA integrity number RIN for each sample. Only RINs ≥8 were accepted for library synthesis. RNA concentration and purity was assessed on a Qubit Fluorometer using the Qubit® RNA HS High Sensitivity Assay Kit according to the manufacturer's instructions. The values obtained for RNA concentration were used to calculate the amount needed for the library preparation. Total RNA was converted to a cDNA library using the Illumina TruSeq® Stranded mRNA Library Preparation Kit following the manufacturer's instructions. An RNA input of 1000 ng was used for larval samples and 600 ng for adult heart samples. The only minor but important deviation from the protocol was a shorter drying time for the magnetic beads due to the temperature of the lab environment. The recommended drying time of 15 minutes was reduced to 10 minutes. Briefly the polyA containing mRNA molecules were purified and fragmented into smaller pieces. First strand cDNA was synthesised from the cleaved RNA fragments using reverse transcriptase and random primers. The RNA fragments were removed and double stranded cDNA was synthesised with the incorporation of dUTP instead of dTTP in the second strand. Strand specificity was achieved by degradation of the second strand as the polymerase used does not recognise past the dUTP nucleotide. The three prime ends of the cDNA fragments were adenylated to prevent them ligating to each other. Complementary indexing adapters were ligated to the ends of the fragments to allow hybridisation of the fragments to the flow cell at the sequencing stage. A final PCR step ensured enrichment of cDNA fragments with adapters at both ends and amplification of the library. The size and purity of each library sample was assessed on an Agilent Bioanalyzer using the Agilent DNA 1000 Kit. The DNA of each library sample was quantified on a Qubit Fluorometer using the Qubit dsDNA HS High Sensitivity Assay Kit according to the manufacturer's instructions. All 12 samples were pooled at equal concentration and the quality of the final product was validated on an Agilent Bioanalyzer. The average fragment size was 270 bp.	WT embryo rep 1	SAMEA5585435	Dept. of Pharmacology and Therapeutics, College of Medicine, Nursing and Heath Sciences, National University of Ireland, Galway	ENA FIRST PUBLIC:2021 04 30T00:25:03Z\|ENA LAST UPDATE:2019 04 30T15:21:47Z\|External Id:SAMEA5585435\|INSDC center name:Dept. of Pharmacology and Therapeutics College of Medicine Nursing and Heath Sciences National University of Ireland Galway\|INSDC first public:2021 04 30T00:25:03Z\|INSDC last update:2019 04 30T15:21:47Z\|INSDC status:public\|Submitter Id:E MTAB 7920:WT embryo rep 1\|age:5\|broker name:ArrayExpress\|common name:zebrafish\|developmental stage:Danio rerio larval stage\|disease:normal\|genotype:wild type genotype\|individual:mixed pool of 10 larvae\|organism part:whole organism\|sample name:E MTAB 7920:WT embryo rep 1\|scientific name:Danio rerio\|strain:AB									NextSeq 500 paired end sequencing; RNA seq of zebrafish sa12692 mutants against WT controls	E MTAB 7920:WT embryo rep 1 p	WT embryo rep 1 p	RNA seq of zebrafish sa12692 mutants against WT controls	Larvae were euthanized by rapid freezing. Ten larvae were pooled per sample and homogenized in TRIzol®. All centrifugation steps for RNA extractions occurred at 4°C. RNA was extracted using the TRIzol® method. Pools of 10 zebrafish larvae per biological replicate were collected at 5 dpf and placed in a 1.5 mL tube. Two adult hearts were pooled per biological replicate; these were transferred from RNAlater® to a 1.5 mL tube. TRIzol® 250 μL was added to each tube. The samples were homogenized thoroughly using a sterile 21G needle and a 1 mL syringe. Samples were incubated for 5 minutes at room temperature RT. Chloroform was added one fifth of the volume of TRIzol® used followed by immediate agitation for 15 seconds and incubation at RT for 3 minutes. For phase separation samples were centrifuged at 12000 g for 20 min. The upper aqueous phase containing the RNA was removed carefully and transferred to a new tube. Isopropanol was added half the volume of TRIzol® used and mixed gently. Samples were incubated for 10 minutes at RT and centrifuged at 18000 g for 10 minutes. The supernatant was removed and the pellet was washed in 70% ice cold ethanol one tenth of the volume of TRIzol® used followed by centrifugation at 18000 g for 10 minutes. The ethanol was removed and the pellet was allowed to dry at RT for 7 minutes. The pellet was resuspended in 88 µL of nuclease free water and heated at 55°C for 7 minutes to fully resuspend the pellet. To remove any remaining DNA 2 µL 4 units of DNase enzyme and 10 µL of 10X DNase buffer were added and the samples were incubated for 10 minutes at 37°C. Phenol 200 µL and 20 µL of sodium acetate were added and the samples were centrifuged at 18000 g for 5 minutes. The upper phase was transferred to a new tube and 200 µL of chloroform:isoamyl alcohol 24:1 was added followed by centrifugation at 18000 g for 5 minutes. The chloroform:isoamyl alcohol step was repeated. The upper phase was transferred to a new tube and 200 µL of isopropanol was added to precipitate the RNA. The samples were incubated at 80°C for 30 minutes followed by centrifugation at 18000 g for 30 minutes. The isopropanol was discarded and 200 µL of ice cold ethanol was added to wash the pellet. The samples were centrifuged for a final 5 minutes at 18000 g. The ethanol was discarded and the pellets were air dried for 5 10 minutes. The samples were resuspended in 20 µL nuclease free water. The RNA was stored at 80°C. The quality/integrity of the RNA was assessed with the use of an Agilent RNA 6000 nano kit on an Agilent Bioanalyzer 2100 by following the manufacturer's instructions. RNA integrity was measured by a software tool on the Agilent Bioanalyzer which calculates an RNA integrity number RIN for each sample. Only RINs ≥8 were accepted for library synthesis. RNA concentration and purity was assessed on a Qubit Fluorometer using the Qubit® RNA HS High Sensitivity Assay Kit according to the manufacturer's instructions. The values obtained for RNA concentration were used to calculate the amount needed for the library preparation. Total RNA was converted to a cDNA library using the Illumina TruSeq® Stranded mRNA Library Preparation Kit following the manufacturer's instructions. An RNA input of 1000 ng was used for larval samples and 600 ng for adult heart samples. The only minor but important deviation from the protocol was a shorter drying time for the magnetic beads due to the temperature of the lab environment. The recommended drying time of 15 minutes was reduced to 10 minutes. Briefly the polyA containing mRNA molecules were purified and fragmented into smaller pieces. First strand cDNA was synthesised from the cleaved RNA fragments using reverse transcriptase and random primers. The RNA fragments were removed and double stranded cDNA was synthesised with the incorporation of dUTP instead of dTTP in the second strand. Strand specificity was achieved by degradation of the second strand as the polymerase used does not recognise past the dUTP nucleotide. The three prime ends of the cDNA fragments were adenylated to prevent them ligating to each other. Complementary indexing adapters were ligated to the ends of the fragments to allow hybridisation of the fragments to the flow cell at the sequencing stage. A final PCR step ensured enrichment of cDNA fragments with adapters at both ends and amplification of the library. The size and purity of each library sample was assessed on an Agilent Bioanalyzer using the Agilent DNA 1000 Kit. The DNA of each library sample was quantified on a Qubit Fluorometer using the Qubit dsDNA HS High Sensitivity Assay Kit according to the manufacturer's instructions. All 12 samples were pooled at equal concentration and the quality of the final product was validated on an Agilent Bioanalyzer. The average fragment size was 270 bp.	Experimental Factor: genotype:wild type genotype\|Experimental Factor: developmental stage:Danio rerio larval stage\|Experimental Factor: organism part:whole organism	RNA-Seq	TRANSCRIPTOMIC	PolyA	PAIRED	ILLUMINA	NextSeq 500		ERP115040	NextSeq 500 paired end sequencing; RNA seq of zebrafish sa12692 mutants against WT controls	ENA FIRST PUBLIC:2021 04 30\|ENA LAST UPDATE:2019 04 30	HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1WTembryo1_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1WTembryo1_2.fq.gz	fastq fastq	7300913760.0	45630711.0	E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1WTembryo1	0:80 1:80	A:1931507380;C:1703881934;G:1712942848;T:1951676424;N:905174	80	80			1931507380	1703881934	1712942848	1951676424	905174	ERX3327071	ERS3389659	ERA1880314	Dept. of Pharmacology and Therapeutics, College of Medicine, Nursing and Heath Sciences, National University of Ireland, Galway\|European Nucleotide Archive	Dept. of Pharmacology and Therapeutics, College of Medicine, Nursing and Heath Sciences, National University of Ireland, Galway\|European Nucleotide Archive	2	0.93831	0.95401	0.09471	0.09526	0.67576	0.67403	0.48092	0.48297	80	80	B	B	biological fallback assumption	illumina	nextseq	3prime	poly_a	trueseq	bulk	bulk	bulk		Ireland	2019-04-30	Larval	Larval	Whole Organism	All anatomical structures