run_metadata: 10309

This data as json

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10309	ERR7179904	ERX6749461	ERS8138767	ERP132743	PRJEB48372	Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos	E-MTAB-11092	Transcriptome Analysis	The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos Danio rerio using transcriptomic profiling. Clobetasol propionate a highly potent synthetic corticosteroid which is known to have immunosuppressive effects on vertebrates including zebrafish was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection the embryos were injected with a mixture of different pathogen associated molecular patterns PAMPs consisting of Pam3CSK4 synthetic lipopeptide polyI:C synthetic viral dsRNA and flagellin isolated from Bacillus subtilis. Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test OECD 236 20 fertilized eggs were exposed to a sub lethal concentration 250 nM of clobetasol propionate for xxx hours under semi static conditions. The test comprised of three treatments with clobetasol propionate and two negative control NC groups and was performed in triplicates. At 46 hpf the eggs were manually dechorinated. At 48 hpf the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection 8 nL of the mixed PAMPs 0.33 µg/mL Pam3CSK4 0.33 µg/mL polyI:C and 0.033 µg/mL flagellin were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. post injection the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit Macherey Nagel. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 4000 System Illumina in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.	ENA FIRST PUBLIC:2022 02 25\|ENA LAST UPDATE:2022 02 25		Protocols: For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2°C on a 12:12 h light/dark cycle. They were fed daily with TetraMin® Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1°C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 µM in acetone before 500 µL were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 µL of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the “growth protocol”. Standard flagellin from Bacillus subtilis ≈ 10% purity Pam3CSK4 ≥ 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturer's instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 µL of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 µL PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 µm. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 µg/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24© MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturer's protocol. RNA concentration >100 ng/µl was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The sample's overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer© Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit© Agilent 5067 1511 according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/µl total RNA at the sequencing facility “NGS Services for Integrative Genomics” at the University of Göttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.	R1287	SAMEA10486022	Fraunhofer Attract Eco'n'OMICs, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany	ENA first public:2022 01 01\|ENA last update:2022 01 01\|External Id:SAMEA10486022\|INSDC center alias:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany\|INSDC center name:Fraunhofer Attract Eco'n'OMICs Fraunhofer Institute for Molecular Biology and Applied Ecology Schmallenberg Germany\|INSDC first public:2022 01 01T00:13:13Z\|INSDC last update:2022 01 01T00:13:13Z\|INSDC status:public\|Submitter Id:E MTAB 11092:R1287\|age:51\|broker name:ArrayExpress\|common name:zebrafish\|developmental stage:hatching long pec\|genotype:wild type genotype\|organism part:whole organism\|replicate:T6\|sample name:E MTAB 11092:R1287\|sex:mixed\|stimulus:PAMP\|strain:AB									Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos	E MTAB 11092:R1287 s	R1287 s	Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos	For each sample 10 zebrafish larvae were randomly picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Once individuals were collected for all samples the supernatant was removed and the tubes were snap frozen in liquid nitrogen. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the Growth protocol. For detailed information about exposure duration and conditions please refer to the Treatment protocol. Adult animal husbandry: Wild type zebrafish Danio rerio Strain AB broodstocks were maintained under flow through conditions in 150 L tanks at 26 +/ 2C on a 12:12 h light/dark cycle. They were fed daily with TetraMin Tetra Werke Melle Germany main feed ad libitum and nauplii of Artemia salina. The day before test start glass spawning trays with artificial substrate green glass beads stringed on stainless steel wire were placed at the bottom of each tank. post mating and spawning in the morning hours eggs were rinsed with clear Cu reduced water and placed into glass dishes for pre picking. Fish embryo incubation: For each sample 20 fertilized eggs in the early blastula stage were placed in glass petri dishes diameter 6 cm filled with 8 mL of the testing solution or control medium. Embryos were incubated at 27 +/ 1C on a 14:10 h light/dark cycle. At 24 hpf eggs were inspected while half of the test solution was renewed. Clobetasol propionate CAS: 25122 46 7 was purchased from Sigma Aldrich Taufkirchen Germany. For the test solution preparation first a 5 mM stock solution was prepared in Acetone 99.9% MS grade. This stock was diluted to 250 M in acetone before 500 L were added to a 500 mL DURAN glass bottle. To the bottle for the control solution only 500 L of pure Acetone was added. post complete evaporation of the acetone the bottle was filled up with 500 mL copper reduced tap water. Complete dissolution of precipitated clobetasol propionate was achieved by sonication for 30 minutes Sonorex RK 100H Bandelin Germany. The test solutions were freshly prepared one day prior the start of the experiment and were constantly aerated at room temperature. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. Per sample about 40 50 fertilized fish eggs were pre picked in glass petri dishes 10 cm diameter filled with 30 ml of the testing solution. Using an optical binokular microscope 20 embryos of the same early blastula stage were transferred from the pre picking plate into the test glass wells filled with 8 ml of the test solution. Embryos were then incubated as described in the growth protocol. Standard flagellin from Bacillus subtilis 10% purity Pam3CSK4 98 % purity and PolyI:C HMW high molecular weight were purchased from InvivoGen SAS Toulouse France. Stock solutions 1 mg/mL for polyI:C and Pam3CSK4; 0.1 mg/mL for flagellin were prepared in provided endotoxin free water aliquoted and stored according to the manufacturers instructions until further use. At approximately 46 hpf the embryos were manually dechorinated and then further incubated. A mixture of the different PAMPs was prepared by mixing 10 L of each component resulting in final concentrations of 0.33 mg/mL for polyI:C and Pam3CSK4 and 0.033 mg/mL for flagellin respectively. The device for microinjection was set up using FemtoTip II injection needles with a complementary universal capillary holder Eppendorf Hamburg Germany connected to an IM 300 microinjector Narishige London United Kingdom. Volume calibration was performed according to Sive et al. 2010 using a micrometer slide with 1/100 mm scale Bresser Rhede Germany. Briefly the needles were backfilled with 10 L PAMP mix or ultra pure water. Injections for volume calibration were made into a drop of immersion oil purred on the micrometer slide the output pressure was constantly kept at 10 psi while the time of injection was adjusted until an injected drop had a diameter of approximately 250 m. Assuming that the drop forms a perfect sphere this corresponds to a volume of about 8 nL. The procedure described below was performed for each of the five conditions in turn treating the three replicates simultaneously. In order to anaesthetize the embryos tricaine Sigma Aldrich Taufkirchen Germany stock solution 4 mg/mL in 21 mM Tris pH 9 was added to the petri dishes to a final concentration of 200 g/mL. The immobilized embryos were transferred onto a 1 % agarose plate which was divided into segments one for each replicate. Excess liquid was removed so that the embryos were kept in place by surface tension. Desiccation was prevented by adding liquid if needed. To ensure that the injections could be performed as quickly as possible the embryos were arranged in a row in a way that their dorsal sides pointed to the same direction. The puncture was made on the dorsal side of the yolk sac and the needle was advanced through the yolk until it reached the yolk sac circulation valley on the ventral side of the embryo where the injection was made directly into the bloodstream. post all replicates of a certain condition were processed the embryos were rinsed back into the corresponding dish containing renewed test solution. Total RNA was extracted using the NucleoSpin RNA/Protein kit from Macherey Nagel 740933.250 The pooled and snap frozen larvae were suspended in RP1 buffer and were transferred to a screw cap tubes filled with 0.3 g of Lysing Matrix D MP Biomedicals 6913050 ceramic beads. Tissue homogenization was performed at 5 m/s for 45 s with FastPrep 24 MP Biomedicals 6004500 at room temperature. From here on RNA was extracted according to the manufacturers protocol. RNA concentration >100 ng/l was measured using a Nanodrop 2000 spectrophotometer Thermo Scientific. The samples overall RNA quality and corresponding RIN value was fluorometrically determined with a 2100 Bioanalyzer Instrument system Agilent G2939BA using the Agilent RNA 6000 Nano Kit Agilent 5067 1511 according to the manufacturers protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at 80C until they were send to the sequencing facility on dry ice. Sequencing libraries were prepared for each sample from 100 ng/l total RNA at the sequencing facility NGS Services for Integrative Genomics at the University of Gttingen in Germany. According to their standard workflow cDNA libraries were prepared from protein coding mRNA that were purified through PolyA selection using the TruSeq RNA Library Prep Kit v2 Illumina. Libraries were validated using a Fragment Analyzer system Agilent Santa Clara USA before sequencing.	Experimental Factor: compound:Clobetasol propionate\|Experimental Factor: dose:250\|Experimental Factor: stimulus:PAMP	RNA-Seq	TRANSCRIPTOMIC	PolyA	SINGLE	ILLUMINA	Illumina HiSeq 4000		ERP132743	Illumina HiSeq 4000 sequencing; Transcriptomic profiling of clobetasol propionate induced immunosuppression in challenged zebrafish embryos	ENA FIRST PUBLIC:2022 02 25\|ENA LAST UPDATE:2022 02 25\|loader:fastq load.py	p946sR1287_sr.fastq.gz	fastq	2134815688.0	42280846.0	E MTAB 11092:R1287	0:50.49	A:565052096;C:502336362;G:482125031;T:582647501;N:2654698	50				565052096	502336362	482125031	582647501	2654698	ERX6749461	ERS8138767	ERA6860152	Fraunhofer Attract Eco	Fraunhofer Attract Eco	1	0.9356		0.11997		0.68314		0.47934		51		B		usable mapping rate	illumina	hiseq_era	unknown	poly_a	trueseq	bulk	unknown	unknown		Germany	2022-01-01	Hatching	Embryo	Whole Organism	All anatomical structures