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
9699,ERR3301006,ERX3327073,ERS3389661,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 3,SAMEA5585437,"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:SAMEA5585437|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 3|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 3|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 3 p,WT embryo rep 3 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_lane1WTembryo3_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1WTembryo3_2.fq.gz,fastq fastq,7308473280.0,45677958.0,E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1WTembryo3 ,0:80 1:80,A:1898812783;C:1731426486;G:1757309247;T:1920031807;N:892957,80,80,,,1898812783,1731426486,1757309247,1920031807,892957,ERX3327073,ERS3389661,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",2,0.94288,0.95671,0.07985,0.08023,0.67915,0.67815,0.47781,0.48461,80,80,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
9700,ERR3301005,ERX3327072,ERS3389660,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 2,SAMEA5585436,"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:SAMEA5585436|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 2|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 2|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 2 p,WT embryo rep 2 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_lane1WTembryo2_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1WTembryo2_2.fq.gz,fastq fastq,7424396000.0,46402475.0,E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1WTembryo2 ,0:80 1:80,A:1940921070;C:1754354124;G:1768688429;T:1959515088;N:917289,80,80,,,1940921070,1754354124,1768688429,1959515088,917289,ERX3327072,ERS3389660,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",2,0.94222,0.95663,0.0909,0.09022,0.67929,0.67606,0.48763,0.48719,80,80,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
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
9705,ERR3301000,ERX3327067,ERS3389655,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.,Mutant embryo rep 3,SAMEA5585431,"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:SAMEA5585431|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:Mutant embryo rep 3|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:Danio rerio larval stage|disease:Naxos disease|genotype:sa12692 mutant|individual:mixed pool of 10 larvae|organism part:whole organism|sample name:E MTAB 7920:Mutant embryo rep 3|scientific name:Danio rerio|strain:sa12692 mutant,,,,,,,,,NextSeq 500 paired end sequencing; RNA seq of zebrafish sa12692 mutants against WT controls,E MTAB 7920:Mutant embryo rep 3 p,Mutant embryo rep 3 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:sa12692 mutant|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_lane1Mutantembryo3_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1Mutantembryo3_2.fq.gz,fastq fastq,6323854560.0,39524091.0,E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1Mutantembryo3 ,0:80 1:80,A:1662088892;C:1473777430;G:1500364334;T:1686853051;N:770853,80,80,,,1662088892,1473777430,1500364334,1686853051,770853,ERX3327067,ERS3389655,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",2,0.93548,0.95404,0.09034,0.09103,0.67584,0.67592,0.47892,0.48193,80,80,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
9706,ERR3300999,ERX3327066,ERS3389654,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.,Mutant embryo rep 2,SAMEA5585430,"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 30T15:21:46Z|External Id:SAMEA5585430|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 30T15:21:46Z|INSDC status:public|Submitter Id:E MTAB 7920:Mutant embryo rep 2|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:Danio rerio larval stage|disease:Naxos disease|genotype:sa12692 mutant|individual:mixed pool of 10 larvae|organism part:whole organism|sample name:E MTAB 7920:Mutant embryo rep 2|scientific name:Danio rerio|strain:sa12692 mutant,,,,,,,,,NextSeq 500 paired end sequencing; RNA seq of zebrafish sa12692 mutants against WT controls,E MTAB 7920:Mutant embryo rep 2 p,Mutant embryo rep 2 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:sa12692 mutant|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_lane1Mutantembryo2_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1Mutantembryo2_2.fq.gz,fastq fastq,6564227680.0,41026423.0,E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1Mutantembryo2 ,0:80 1:80,A:1702331829;C:1557343680;G:1583770440;T:1719974488;N:807243,80,80,,,1702331829,1557343680,1583770440,1719974488,807243,ERX3327066,ERS3389654,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",2,0.94141,0.95836,0.07518,0.07501,0.67982,0.67803,0.47895,0.47763,80,80,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
9707,ERR3300998,ERX3327065,ERS3389653,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.,Mutant embryo rep 1,SAMEA5585429,"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 30T15:21:46Z|External Id:SAMEA5585429|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 30T15:21:46Z|INSDC status:public|Submitter Id:E MTAB 7920:Mutant embryo rep 1|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:Danio rerio larval stage|disease:Naxos disease|genotype:sa12692 mutant|individual:mixed pool of 10 larvae|organism part:whole organism|sample name:E MTAB 7920:Mutant embryo rep 1|scientific name:Danio rerio|strain:sa12692 mutant,,,,,,,,,NextSeq 500 paired end sequencing; RNA seq of zebrafish sa12692 mutants against WT controls,E MTAB 7920:Mutant embryo rep 1 p,Mutant 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:sa12692 mutant|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_lane1Mutantembryo1_1.fq.gz HJGMYBGX2_Zebrafish_mRNA_17s001029_1_1_Morris_lane1Mutantembryo1_2.fq.gz,fastq fastq,6991429280.0,43696433.0,E MTAB 7920:HJGMYBGX2 Zebrafish mRNA 17s001029 1 1 Morris lane1Mutantembryo1 ,0:80 1:80,A:1852543850;C:1630880197;G:1635963943;T:1871173669;N:867621,80,80,,,1852543850,1630880197,1635963943,1871173669,867621,ERX3327065,ERS3389653,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",2,0.93819,0.95201,0.10106,0.10107,0.67541,0.67523,0.4792,0.48253,80,80,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
11226,ERR10368639,ERX9900562,ERS13563108,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X9,SAMEA111469072,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469072|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X9|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:snrnp70  / |organism part:whole organism|sample name:E MTAB 12301:16261X9,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X9 p,16261X9 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:snrnp70  / ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X9_190815_A00421_0101_BHFL23DRXX_S2_L001_R1_001.fastq.gz 16261X9_190815_A00421_0101_BHFL23DRXX_S2_L001_R2_001.fastq.gz,fastq fastq,2686590240.0,26339120.0,E MTAB 12301:16261X9 190815 A00421 0101 BHFL23DRXX S2 L001 R,0:51 1:51,A:694597606;C:635153638;G:628248232;T:728333026;N:257738,51,51,,,694597606,635153638,628248232,728333026,257738,ERX9900562,ERS13563108,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94614,0.94723,0.1538,0.15109,0.70398,0.70352,0.48123,0.48168,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11227,ERR10368638,ERX9900561,ERS13563107,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X8,SAMEA111469071,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469071|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X8|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:snrnp70  / |organism part:whole organism|sample name:E MTAB 12301:16261X8,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X8 p,16261X8 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:snrnp70  / ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X8_190815_A00421_0101_BHFL23DRXX_S4_L001_R1_001.fastq.gz 16261X8_190815_A00421_0101_BHFL23DRXX_S4_L001_R2_001.fastq.gz,fastq fastq,15003930006.0,147097353.0,E MTAB 12301:16261X8 190815 A00421 0101 BHFL23DRXX S4 L001 R,0:51 1:51,A:3882939949;C:3570150556;G:3520942177;T:4028462946;N:1434378,51,51,,,3882939949,3570150556,3520942177,4028462946,1434378,ERX9900561,ERS13563107,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94373,0.94762,0.17954,0.17832,0.70299,0.70207,0.48822,0.48842,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11228,ERR10368637,ERX9900560,ERS13563106,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X7,SAMEA111469070,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469070|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X7|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:snrnp70  / |organism part:whole organism|sample name:E MTAB 12301:16261X7,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X7 p,16261X7 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:snrnp70  / ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|loader:fastq load.py,16261X7_190815_A00421_0101_BHFL23DRXX_S6_L001_R1_001.fastq.gz 16261X7_190815_A00421_0101_BHFL23DRXX_S6_L001_R2_001.fastq.gz,fastq fastq,3806643060.0,37320030.0,E MTAB 12301:16261X7 190815 A00421 0101 BHFL23DRXX S6 L001 R,0:51 1:51,A:971582421;C:919313664;G:905728446;T:1009653865;N:364664,51,51,,,971582421,919313664,905728446,1009653865,364664,ERX9900560,ERS13563106,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94035,0.94356,0.1661,0.16393,0.70136,0.70082,0.48294,0.48049,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11229,ERR10368636,ERX9900559,ERS13563105,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X6,SAMEA111469069,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469069|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X6|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:+/cyt hSNRNP70|organism part:whole organism|sample name:E MTAB 12301:16261X6,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X6 p,16261X6 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:+/cyt hSNRNP70,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X6_190815_A00421_0101_BHFL23DRXX_S7_L001_R1_001.fastq.gz 16261X6_190815_A00421_0101_BHFL23DRXX_S7_L001_R2_001.fastq.gz,fastq fastq,2875874598.0,28194849.0,E MTAB 12301:16261X6 190815 A00421 0101 BHFL23DRXX S7 L001 R,0:51 1:51,A:745870043;C:683782229;G:673037324;T:772909720;N:275282,51,51,,,745870043,683782229,673037324,772909720,275282,ERX9900559,ERS13563105,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.9435,0.94737,0.16987,0.16888,0.6983,0.69826,0.47122,0.47409,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11230,ERR10368635,ERX9900558,ERS13563104,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X5,SAMEA111469068,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469068|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X5|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:+/cyt hSNRNP70|organism part:whole organism|sample name:E MTAB 12301:16261X5,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X5 p,16261X5 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:+/cyt hSNRNP70,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X5_190815_A00421_0101_BHFL23DRXX_S8_L001_R1_001.fastq.gz 16261X5_190815_A00421_0101_BHFL23DRXX_S8_L001_R2_001.fastq.gz,fastq fastq,2656716072.0,26046236.0,E MTAB 12301:16261X5 190815 A00421 0101 BHFL23DRXX S8 L001 R,0:51 1:51,A:693979046;C:627484571;G:617505105;T:717493108;N:254242,51,51,,,693979046,627484571,617505105,717493108,254242,ERX9900558,ERS13563104,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94387,0.94796,0.16299,0.16183,0.69556,0.69581,0.46842,0.47334,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11231,ERR10368634,ERX9900557,ERS13563103,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X4,SAMEA111469067,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469067|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X4|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:+/cyt hSNRNP70|organism part:whole organism|sample name:E MTAB 12301:16261X4,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X4 p,16261X4 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:+/cyt hSNRNP70,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X4_190815_A00421_0101_BHFL23DRXX_S9_L001_R1_001.fastq.gz 16261X4_190815_A00421_0101_BHFL23DRXX_S9_L001_R2_001.fastq.gz,fastq fastq,2693177298.0,26403699.0,E MTAB 12301:16261X4 190815 A00421 0101 BHFL23DRXX S9 L001 R,0:51 1:51,A:698547378;C:640687214;G:628869192;T:724816757;N:256757,51,51,,,698547378,640687214,628869192,724816757,256757,ERX9900557,ERS13563103,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94101,0.94526,0.17344,0.17201,0.68619,0.68513,0.47366,0.4702,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11232,ERR10368633,ERX9900556,ERS13563102,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X3,SAMEA111469066,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469066|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X3|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:wild type genotype|organism part:whole organism|sample name:E MTAB 12301:16261X3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X3 p,16261X3 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|loader:fastq load.py|options:  dnus,16261X3_190815_A00421_0101_BHFL23DRXX_S10_L001_R1_001.fastq.gz 16261X3_190815_A00421_0101_BHFL23DRXX_S10_L001_R2_001.fastq.gz,fastq fastq,3104796462.0,60878362.0,E MTAB 12301:16261X3 190815 A00421 0101 BHFL23DRXX S10 L001 R,0:51,A:803595677;C:737898133;G:731641815;T:831364716;N:296121,51,,,,803595677,737898133,731641815,831364716,296121,ERX9900556,ERS13563102,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,1,0.94361,,0.16161,,0.69822,,0.4647,,51,,B,,usable mapping rate,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11233,ERR10368632,ERX9900555,ERS13563101,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X2,SAMEA111469065,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469065|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X2|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:wild type genotype|organism part:whole organism|sample name:E MTAB 12301:16261X2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X2 p,16261X2 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X2_190815_A00421_0101_BHFL23DRXX_S11_L001_R1_001.fastq.gz 16261X2_190815_A00421_0101_BHFL23DRXX_S11_L001_R2_001.fastq.gz,fastq fastq,2935625892.0,28780646.0,E MTAB 12301:16261X2 190815 A00421 0101 BHFL23DRXX S11 L001 R,0:51 1:51,A:759844700;C:699591057;G:694530268;T:781380766;N:279101,51,51,,,759844700,699591057,694530268,781380766,279101,ERX9900555,ERS13563101,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.93297,0.93744,0.16748,0.16566,0.69929,0.6996,0.4788,0.47875,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11234,ERR10368631,ERX9900554,ERS13563100,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X12,SAMEA111469064,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469064|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:52Z|INSDC last update:2022 10 31T00:16:52Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X12|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:cyt hSNRNP70/ |organism part:whole organism|sample name:E MTAB 12301:16261X12,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X12 p,16261X12 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:cyt hSNRNP70/ ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X12_190815_A00421_0101_BHFL23DRXX_S3_L001_R1_001.fastq.gz 16261X12_190815_A00421_0101_BHFL23DRXX_S3_L001_R2_001.fastq.gz,fastq fastq,7451469240.0,73053620.0,E MTAB 12301:16261X12 190815 A00421 0101 BHFL23DRXX S3 L001 R,0:51 1:51,A:1950856364;C:1747213644;G:1718730947;T:2033957057;N:711228,51,51,,,1950856364,1747213644,1718730947,2033957057,711228,ERX9900554,ERS13563100,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.9372,0.94118,0.20267,0.19972,0.69794,0.69735,0.47505,0.4883,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11235,ERR10368630,ERX9900553,ERS13563099,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X11,SAMEA111469063,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469063|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:51Z|INSDC last update:2022 10 31T00:16:51Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X11|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:cyt hSNRNP70/ |organism part:whole organism|sample name:E MTAB 12301:16261X11,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X11 p,16261X11 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:cyt hSNRNP70/ ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,16261X11_190815_A00421_0101_BHFL23DRXX_S5_L001_R1_001.fastq.gz 16261X11_190815_A00421_0101_BHFL23DRXX_S5_L001_R2_001.fastq.gz,fastq fastq,2754589458.0,27005779.0,E MTAB 12301:16261X11 190815 A00421 0101 BHFL23DRXX S5 L001 R,0:51 1:51,A:715675810;C:652231961;G:640938288;T:745480460;N:262939,51,51,,,715675810,652231961,640938288,745480460,262939,ERX9900553,ERS13563099,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.93802,0.94247,0.17937,0.17728,0.70195,0.70067,0.48458,0.48364,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11236,ERR10368629,ERX9900552,ERS13563098,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X10,SAMEA111469062,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469062|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:51Z|INSDC last update:2022 10 31T00:16:51Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X10|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:cyt hSNRNP70/ |organism part:whole organism|sample name:E MTAB 12301:16261X10,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X10 p,16261X10 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:cyt hSNRNP70/ ,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|loader:fastq load.py,16261X10_190815_A00421_0101_BHFL23DRXX_S1_L001_R1_001.fastq.gz 16261X10_190815_A00421_0101_BHFL23DRXX_S1_L001_R2_001.fastq.gz,fastq fastq,2319764682.0,22742791.0,E MTAB 12301:16261X10 190815 A00421 0101 BHFL23DRXX S1 L001 R,0:51 1:51,A:596937200;C:555502273;G:546190804;T:620912915;N:221490,51,51,,,596937200,555502273,546190804,620912915,221490,ERX9900552,ERS13563098,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94034,0.94451,0.1621,0.16043,0.69406,0.6928,0.4936,0.49757,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
11237,ERR10368628,ERX9900551,ERS13563097,ERP141667,PRJEB56699,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E-MTAB-12301,Other,It has been recently shown that SNRNP70  a major component of the spliceosome  as well as other splicing regulators are found in axons. To investigate the role of SNRNP70 in axons  we generated a zebrafish null mutant and found motor connectivity defects that can be partially rescued upon transgenic overexpression of cytoplasmic only human SNRNP70 cyt hSNRNP70. To understand the molecular function of the cytoplasmic pool of this splicing protein  we performed this RNA seq experiment with the aim to identify mRNA transcripts whose expression is regulated by the cytoplasmic pool of SNRNP70. To do that  we crossed heterozygous mutant animals that are also positive for the cyt SNRNP70 transgene. We then split embryos into four groups: i sibling  ii siblings/cyt hSNRNP70  iii null  iv null/cyt hSNRNP70. Total RNA was extracted from each one of the four groups three biological replicates per sample and sequenced.,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31,,Protocols: Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,16261X1,SAMEA111469061,Department of Life Sciences University of Bath,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|External Id:SAMEA111469061|INSDC center alias:Department of Life Sciences University of Bath|INSDC center name:Department of Life Sciences University of Bath|INSDC first public:2022 10 31T00:16:51Z|INSDC last update:2022 10 31T00:16:51Z|INSDC status:public|Submitter Id:E MTAB 12301:16261X1|age:28|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|disease:normal|genotype:wild type genotype|organism part:whole organism|sample name:E MTAB 12301:16261X1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,E MTAB 12301:16261X1 p,16261X1 p,RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,Embryos were collected and raised in Danieau's solution together with 2 μΜ 4 OHΤ. Treated embryos were screened for GFP fluorescence at 24hpf and sorted into the four genotypes. Total RNA from whole embryos was isolated from each genotype group at 28hpf using a RNeasy Mini kit and eluted in nuclease free water. Total RNA was purified using Illumina TruSeq Stranded Total RNA Library Prep Ribo Zero Gold,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP141667,Illumina NovaSeq 6000 paired end sequencing; RNA seq of snrnp70 mutant zebrafish and its rescue by overexpression of cytoplasmic SNRNP70,ENA FIRST PUBLIC:2022 10 31|ENA LAST UPDATE:2022 10 31|loader:fastq load.py,16261X1_190815_A00421_0101_BHFL23DRXX_S12_L001_R1_001.fastq.gz 16261X1_190815_A00421_0101_BHFL23DRXX_S12_L001_R2_001.fastq.gz,fastq fastq,2992675512.0,29339956.0,E MTAB 12301:16261X1 190815 A00421 0101 BHFL23DRXX S12 L001 R,0:51 1:51,A:768607745;C:718646844;G:713246748;T:791888447;N:285728,51,51,,,768607745,718646844,713246748,791888447,285728,ERX9900551,ERS13563097,ERA18523376,Department of Life Sciences University of Bath|European Nucleotide Archive,Department of Life Sciences University of Bath|European Nucleotide Archive,2,0.94414,0.94881,0.16725,0.16648,0.68909,0.68923,0.47873,0.48244,51,51,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2022-10-31,Pharyngula,Embryo,Whole Organism,All anatomical structures
15394,ERR12724517,ERX12099016,ERS18400121,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 WT 2,E MTAB 13886:Foxg1 WT 2,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a WT|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 WT 2 p,Foxg1 WT 2 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X14_200629_A00421_0211_BHN23CDRXX_S26_L001_R1_001.fastq.gz 18067X14_200629_A00421_0211_BHN23CDRXX_S26_L001_R2_001.fastq.gz,fastq fastq,4054926360.0,39754180.0,E MTAB 13886:18067X14 200629 A00421 0211 BHN23CDRXX S26 L001 R,0:51 1:51,A:1027112898;C:978274968;G:992295226;T:1057002684;N:240584,51,51,,,1027112898,978274968,992295226,1057002684,240584,ERX12099016,ERS18400121,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15395,ERR12724511,ERX12099010,ERS18400115,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 9,E MTAB 13886:Foxg1 het 9,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 9 p,Foxg1 het 9 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X11_200629_A00421_0211_BHN23CDRXX_S18_L001_R1_001.fastq.gz 18067X11_200629_A00421_0211_BHN23CDRXX_S18_L001_R2_001.fastq.gz,fastq fastq,2866917570.0,28107035.0,E MTAB 13886:18067X11 200629 A00421 0211 BHN23CDRXX S18 L001 R,0:51 1:51,A:725353172;C:688761995;G:699694713;T:752936948;N:170742,51,51,,,725353172,688761995,699694713,752936948,170742,ERX12099010,ERS18400115,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15396,ERR12724513,ERX12099012,ERS18400117,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 null 2,E MTAB 13886:Foxg1 null 2,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a hom|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 null 2 p,Foxg1 null 2 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X2_200629_A00421_0211_BHN23CDRXX_S27_L001_R1_001.fastq.gz 18067X2_200629_A00421_0211_BHN23CDRXX_S27_L001_R2_001.fastq.gz,fastq fastq,2908778880.0,28517440.0,E MTAB 13886:18067X2 200629 A00421 0211 BHN23CDRXX S27 L001 R,0:51 1:51,A:734785178;C:700849624;G:712519278;T:760452796;N:172004,51,51,,,734785178,700849624,712519278,760452796,172004,ERX12099012,ERS18400117,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15397,ERR12724507,ERX12099006,ERS18400111,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 5,E MTAB 13886:Foxg1 het 5,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 5 p,Foxg1 het 5 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X7_200629_A00421_0211_BHN23CDRXX_S19_L001_R1_001.fastq.gz 18067X7_200629_A00421_0211_BHN23CDRXX_S19_L001_R2_001.fastq.gz,fastq fastq,3111366282.0,30503591.0,E MTAB 13886:18067X7 200629 A00421 0211 BHN23CDRXX S19 L001 R,0:51 1:51,A:780094412;C:754603273;G:768441715;T:808041650;N:185232,51,51,,,780094412,754603273,768441715,808041650,185232,ERX12099006,ERS18400111,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15398,ERR12724519,ERX12099018,ERS18400123,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 WT 6,E MTAB 13886:Foxg1 WT 6,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a WT|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 WT 6 p,Foxg1 WT 6 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X16_200629_A00421_0211_BHN23CDRXX_S22_L001_R1_001.fastq.gz 18067X16_200629_A00421_0211_BHN23CDRXX_S22_L001_R2_001.fastq.gz,fastq fastq,3030554130.0,29711315.0,E MTAB 13886:18067X16 200629 A00421 0211 BHN23CDRXX S22 L001 R,0:51 1:51,A:770513445;C:725582256;G:737052763;T:797224111;N:181555,51,51,,,770513445,725582256,737052763,797224111,181555,ERX12099018,ERS18400123,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15399,ERR12724510,ERX12099009,ERS18400114,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 8,E MTAB 13886:Foxg1 het 8,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 8 p,Foxg1 het 8 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X10_200629_A00421_0211_BHN23CDRXX_S13_L001_R1_001.fastq.gz 18067X10_200629_A00421_0211_BHN23CDRXX_S13_L001_R2_001.fastq.gz,fastq fastq,3601017894.0,35304097.0,E MTAB 13886:18067X10 200629 A00421 0211 BHN23CDRXX S13 L001 R,0:51 1:51,A:903986531;C:871550198;G:885398218;T:939870481;N:212466,51,51,,,903986531,871550198,885398218,939870481,212466,ERX12099009,ERS18400114,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15400,ERR12724516,ERX12099015,ERS18400120,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 WT 1,E MTAB 13886:Foxg1 WT 1,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a WT|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 WT 1 p,Foxg1 WT 1 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X13_200629_A00421_0211_BHN23CDRXX_S14_L001_R1_001.fastq.gz 18067X13_200629_A00421_0211_BHN23CDRXX_S14_L001_R2_001.fastq.gz,fastq fastq,4252867662.0,41694781.0,E MTAB 13886:18067X13 200629 A00421 0211 BHN23CDRXX S14 L001 R,0:51 1:51,A:1069523245;C:1031533375;G:1047047746;T:1104513602;N:249694,51,51,,,1069523245,1031533375,1047047746,1104513602,249694,ERX12099015,ERS18400120,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15401,ERR12724505,ERX12099004,ERS18400109,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 2,E MTAB 13886:Foxg1 het 2,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 2 p,Foxg1 het 2 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X5_200629_A00421_0211_BHN23CDRXX_S21_L001_R1_001.fastq.gz 18067X5_200629_A00421_0211_BHN23CDRXX_S21_L001_R2_001.fastq.gz,fastq fastq,3490870236.0,34224218.0,E MTAB 13886:18067X5 200629 A00421 0211 BHN23CDRXX S21 L001 R,0:51 1:51,A:868722737;C:853509703;G:868564192;T:899868487;N:205117,51,51,,,868722737,853509703,868564192,899868487,205117,ERX12099004,ERS18400109,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15402,ERR12724509,ERX12099008,ERS18400113,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 7,E MTAB 13886:Foxg1 het 7,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 7 p,Foxg1 het 7 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X9_200629_A00421_0211_BHN23CDRXX_S15_L001_R1_001.fastq.gz 18067X9_200629_A00421_0211_BHN23CDRXX_S15_L001_R2_001.fastq.gz,fastq fastq,3288698484.0,32242142.0,E MTAB 13886:18067X9 200629 A00421 0211 BHN23CDRXX S15 L001 R,0:51 1:51,A:849623075;C:775307492;G:786016403;T:877557952;N:193562,51,51,,,849623075,775307492,786016403,877557952,193562,ERX12099008,ERS18400113,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15403,ERR12724512,ERX12099011,ERS18400116,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 null 1,E MTAB 13886:Foxg1 null 1,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a hom|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 null 1 p,Foxg1 null 1 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X1_200629_A00421_0211_BHN23CDRXX_S28_L001_R1_001.fastq.gz 18067X1_200629_A00421_0211_BHN23CDRXX_S28_L001_R2_001.fastq.gz,fastq fastq,3735665544.0,36624172.0,E MTAB 13886:18067X1 200629 A00421 0211 BHN23CDRXX S28 L001 R,0:51 1:51,A:930463361;C:914183345;G:929494372;T:961303244;N:221222,51,51,,,930463361,914183345,929494372,961303244,221222,ERX12099011,ERS18400116,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15404,ERR12724514,ERX12099013,ERS18400118,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 null 3,E MTAB 13886:Foxg1 null 3,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a hom|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 null 3 p,Foxg1 null 3 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X3_200629_A00421_0211_BHN23CDRXX_S25_L001_R1_001.fastq.gz 18067X3_200629_A00421_0211_BHN23CDRXX_S25_L001_R2_001.fastq.gz,fastq fastq,3581623512.0,35113956.0,E MTAB 13886:18067X3 200629 A00421 0211 BHN23CDRXX S25 L001 R,0:51 1:51,A:901782474;C:866330420;G:882309046;T:930988392;N:213180,51,51,,,901782474,866330420,882309046,930988392,213180,ERX12099013,ERS18400118,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15405,ERR12724504,ERX12099003,ERS18400108,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 13,E MTAB 13886:Foxg1 het 13,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 13 p,Foxg1 het 13 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X12_200629_A00421_0211_BHN23CDRXX_S16_L001_R1_001.fastq.gz 18067X12_200629_A00421_0211_BHN23CDRXX_S16_L001_R2_001.fastq.gz,fastq fastq,3198417570.0,31357035.0,E MTAB 13886:18067X12 200629 A00421 0211 BHN23CDRXX S16 L001 R,0:51 1:51,A:806440222;C:774073728;G:785492238;T:832222984;N:188398,51,51,,,806440222,774073728,785492238,832222984,188398,ERX12099003,ERS18400108,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15406,ERR12724508,ERX12099007,ERS18400112,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 6,E MTAB 13886:Foxg1 het 6,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 6 p,Foxg1 het 6 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X8_200629_A00421_0211_BHN23CDRXX_S17_L001_R1_001.fastq.gz 18067X8_200629_A00421_0211_BHN23CDRXX_S17_L001_R2_001.fastq.gz,fastq fastq,2829840366.0,27743533.0,E MTAB 13886:18067X8 200629 A00421 0211 BHN23CDRXX S17 L001 R,0:51 1:51,A:712954704;C:683604864;G:696251651;T:736863084;N:166063,51,51,,,712954704,683604864,696251651,736863084,166063,ERX12099007,ERS18400112,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15407,ERR12724506,ERX12099005,ERS18400110,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 het 3,E MTAB 13886:Foxg1 het 3,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a het|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 het 3 p,Foxg1 het 3 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X6_200629_A00421_0211_BHN23CDRXX_S20_L001_R1_001.fastq.gz 18067X6_200629_A00421_0211_BHN23CDRXX_S20_L001_R2_001.fastq.gz,fastq fastq,2947992168.0,28901884.0,E MTAB 13886:18067X6 200629 A00421 0211 BHN23CDRXX S20 L001 R,0:51 1:51,A:745555818;C:709342231;G:720902928;T:772016275;N:174916,51,51,,,745555818,709342231,720902928,772016275,174916,ERX12099005,ERS18400110,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15408,ERR12724515,ERX12099014,ERS18400119,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 null 6,E MTAB 13886:Foxg1 null 6,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a hom|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 null 6 p,Foxg1 null 6 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X4_200629_A00421_0211_BHN23CDRXX_S23_L001_R1_001.fastq.gz 18067X4_200629_A00421_0211_BHN23CDRXX_S23_L001_R2_001.fastq.gz,fastq fastq,3166535838.0,31044469.0,E MTAB 13886:18067X4 200629 A00421 0211 BHN23CDRXX S23 L001 R,0:51 1:51,A:781063082;C:780685132;G:798403519;T:806197923;N:186182,51,51,,,781063082,780685132,798403519,806197923,186182,ERX12099014,ERS18400119,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
15409,ERR12724518,ERX12099017,ERS18400122,ERP158370,PRJEB73599,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E-MTAB-13886,Transcriptome Analysis,Bulk tissue RNA sequencing of individual 24hpf zebrafish larvae to compare the gene expression values between wild type and foxg1a nonsense mutants heterozygous and homozygous mutants.   The mutation is a 5bp deletion 32bp from canonical start codon; AAATG deleted.,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,,Protocols: Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,Foxg1 WT 5,E MTAB 13886:Foxg1 WT 5,,isolate:not applicable|organism:Danio rerio|organism:Danio rerio|collection date:not collected|scientific name:Danio rerio|common name:zebrafish|organism part:whole organism|developmental stage:pharyngula prim 5|genotype:Foxg1a WT|geographic location country and/or sea:not collected,,,,,,,,,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,E MTAB 13886:Foxg1 WT 5 p,Foxg1 WT 5 p,RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,Heterozygous mutant 5bp deletion 32 bp from canonical start codon; AAATG foxg1a adult zebrafish in a dlx5 6;GFP background were incrossed and progeny collected. Dlx+ interneuron phenotype was used to identify homozygous no interneurons in telencephalon and heterozygous reduced number of interneurons in telencephalon. Genotyping was confirmed with RNA sequencing reads at the foxg1a gene locus to ensure correct genotyping. RNA was extracted from single 24hpf zebrafish using the Qiagen RNeasy Micro Kit Qiagen  74004. RNA concentration was measured using the Qubit reagent kit and a small sample was tested for RNA integrity using a bioanalyzer instrument. Only samples with an RNA integrity number of 9 or above were used in this experiment. Total RNA Library was repapred using the Illumina TruSeq Stranded Total RNA Library kit. Ribosomal RNAs were depleted using Ribo Zero Gold.,,RNA-Seq,TRANSCRIPTOMIC,Inverse rRNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP158370,Illumina NovaSeq 6000 paired end sequencing; RNA seq of 24hpf Zebrafish larvae comparing foxg1a mutants to WT siblings,ENA FIRST PUBLIC:2024 03 29|ENA LAST UPDATE:2024 03 29,18067X15_200629_A00421_0211_BHN23CDRXX_S24_L001_R1_001.fastq.gz 18067X15_200629_A00421_0211_BHN23CDRXX_S24_L001_R2_001.fastq.gz,fastq fastq,3253306932.0,31895166.0,E MTAB 13886:18067X15 200629 A00421 0211 BHN23CDRXX S24 L001 R,0:51 1:51,A:822636212;C:784647591;G:797221431;T:848609081;N:192617,51,51,,,822636212,784647591,797221431,848609081,192617,ERX12099017,ERS18400122,ERA29264914,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,rrna_depletion,trueseq,bulk,bulk,bulk,,United Kingdom,2024-03-29,Pharyngula,Embryo,Whole Organism,All anatomical structures
24652,SRR25487068,SRX21218619,SRS18475798,SRP452670,PRJNA1000968,CRISPR/Cas9 mediated Nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo,GSE239788,Transcriptome Analysis,Nexilin NEXN plays a crucial role in stabilizing the sarcomeric Z disk of striated muscle fibers and  when mutated   leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice  the detailed impact of the  constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated.  We characterized a constitutive homozygous CRISPR/Cas9 mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired  skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably  in contrast to nexn  morphants  CRISPR/Cas9 nexn /  knockout embryos showed a milder phenotype without xxx development of a pronounced  pericardial edema or blood congestion. nexn specific expression analysis as well as whole transcriptome profiling suggest  some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced  and may mediate a sarcomere stabilizing function in nexn /  knockout embryos. Overall design: To investigate the influence of nexn knockout on cardiac and skeletal muslce  we generated a CRISPR/Cas9 mediated nexn  knockout zebrafish model. We then performed gene expression profiling analysis using data obtained from RNA seq.,,pubmed:38114601,,nexn /   biological replicate 2,GSM7673294,,source name:whole organism|tissue:whole organism|genotype:nexn knockout|geo loc name:missing|collection date:missing,nexn /   biological replicate 2,Raw sequencing data is screened for reads originating from rRNA using RiboDetector eurofins genomics  INVIEW transcriptome High quality sequence reads are aligned to the reference genome using STAR Spliced Transcripts Alignment to a Reference  run through Sentieon framework  along with the known gene models. eurofins genomics  INVIEW transcriptome Gene wise quantification is achieved by inspecting transcriptome alignments using RSEM tool. eurofins genomics  INVIEW transcriptome Assembly: GRCz11 Supplementary files format and content: Sample wise  gene wise read counts  TPM value and FPKM value,whole organism,,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,tissue:whole organism|genotype:nexn knockout,GSM7673294,GSM7673294: nexn /   biological replicate 2; Danio rerio; RNA Seq,GSM7673294 r1,GSM7673294,1,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP452670,,loader:fastq load.py,NG-33220_nexn_E2_mut3_lib700540_10254_1_2.fastq.gz NG-33220_nexn_E2_mut3_lib700540_10254_1_1.fastq.gz,fastq fastq,12821226686.0,42454393.0,GSM7673294 r1,0:151 1:151,A:3460123169;C:2958819646;G:3023309404;T:3378893399;N:81068,151,151,,,3460123169,2958819646,3023309404,3378893399,81068,SRX21218619,SRS18475798,SRA1684694,"Molecular Cardiology, Internal Medicine II, Uniklinik Ulm","Molecular Cardiology, Internal Medicine II, Uniklinik Ulm",2,0.96544,0.96684,0.07136,0.06827,0.66969,0.66914,0.44957,0.45604,151,151,B,B,biological fallback assumption,illumina,novaseq_era,full_length,random_priming,unknown,bulk,bulk,bulk,,Germany,2023-08-01,Undetermined,Embryo,Whole Organism,All anatomical structures
24653,SRR25487069,SRX21218618,SRS18475797,SRP452670,PRJNA1000968,CRISPR/Cas9 mediated Nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo,GSE239788,Transcriptome Analysis,Nexilin NEXN plays a crucial role in stabilizing the sarcomeric Z disk of striated muscle fibers and  when mutated   leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice  the detailed impact of the  constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated.  We characterized a constitutive homozygous CRISPR/Cas9 mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired  skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably  in contrast to nexn  morphants  CRISPR/Cas9 nexn /  knockout embryos showed a milder phenotype without xxx development of a pronounced  pericardial edema or blood congestion. nexn specific expression analysis as well as whole transcriptome profiling suggest  some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced  and may mediate a sarcomere stabilizing function in nexn /  knockout embryos. Overall design: To investigate the influence of nexn knockout on cardiac and skeletal muslce  we generated a CRISPR/Cas9 mediated nexn  knockout zebrafish model. We then performed gene expression profiling analysis using data obtained from RNA seq.,,pubmed:38114601,,nexn /   biological replicate 1,GSM7673293,,source name:whole organism|tissue:whole organism|genotype:nexn knockout|geo loc name:missing|collection date:missing,nexn /   biological replicate 1,Raw sequencing data is screened for reads originating from rRNA using RiboDetector eurofins genomics  INVIEW transcriptome High quality sequence reads are aligned to the reference genome using STAR Spliced Transcripts Alignment to a Reference  run through Sentieon framework  along with the known gene models. eurofins genomics  INVIEW transcriptome Gene wise quantification is achieved by inspecting transcriptome alignments using RSEM tool. eurofins genomics  INVIEW transcriptome Assembly: GRCz11 Supplementary files format and content: Sample wise  gene wise read counts  TPM value and FPKM value,whole organism,,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,tissue:whole organism|genotype:nexn knockout,GSM7673293,GSM7673293: nexn /   biological replicate 1; Danio rerio; RNA Seq,GSM7673293 r1,GSM7673293,1,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP452670,,loader:fastq load.py,NG-33220_nexn_E2_mut1_lib691942_10222_3_2.fastq.gz NG-33220_nexn_E2_mut1_lib691942_10222_3_1.fastq.gz,fastq fastq,6518650974.0,21584937.0,GSM7673293 r1,0:151 1:151,A:1776227735;C:1486881610;G:1503672330;T:1751641678;N:227621,151,151,,,1776227735,1486881610,1503672330,1751641678,227621,SRX21218618,SRS18475797,SRA1684694,"Molecular Cardiology, Internal Medicine II, Uniklinik Ulm","Molecular Cardiology, Internal Medicine II, Uniklinik Ulm",2,0.9619,0.96365,0.08223,0.07902,0.66967,0.66811,0.44763,0.45022,151,151,B,B,biological fallback assumption,illumina,novaseq_era,full_length,random_priming,unknown,bulk,bulk,bulk,,Germany,2023-08-01,Undetermined,Embryo,Whole Organism,All anatomical structures
24654,SRR25487070,SRX21218617,SRS18475796,SRP452670,PRJNA1000968,CRISPR/Cas9 mediated Nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo,GSE239788,Transcriptome Analysis,Nexilin NEXN plays a crucial role in stabilizing the sarcomeric Z disk of striated muscle fibers and  when mutated   leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice  the detailed impact of the  constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated.  We characterized a constitutive homozygous CRISPR/Cas9 mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired  skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably  in contrast to nexn  morphants  CRISPR/Cas9 nexn /  knockout embryos showed a milder phenotype without xxx development of a pronounced  pericardial edema or blood congestion. nexn specific expression analysis as well as whole transcriptome profiling suggest  some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced  and may mediate a sarcomere stabilizing function in nexn /  knockout embryos. Overall design: To investigate the influence of nexn knockout on cardiac and skeletal muslce  we generated a CRISPR/Cas9 mediated nexn  knockout zebrafish model. We then performed gene expression profiling analysis using data obtained from RNA seq.,,pubmed:38114601,,nexn+/+  biological replicate 2,GSM7673292,,source name:whole organism|tissue:whole organism|genotype:WT|geo loc name:missing|collection date:missing,nexn+/+  biological replicate 2,Raw sequencing data is screened for reads originating from rRNA using RiboDetector eurofins genomics  INVIEW transcriptome High quality sequence reads are aligned to the reference genome using STAR Spliced Transcripts Alignment to a Reference  run through Sentieon framework  along with the known gene models. eurofins genomics  INVIEW transcriptome Gene wise quantification is achieved by inspecting transcriptome alignments using RSEM tool. eurofins genomics  INVIEW transcriptome Assembly: GRCz11 Supplementary files format and content: Sample wise  gene wise read counts  TPM value and FPKM value,whole organism,,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,tissue:whole organism|genotype:WT,GSM7673292,GSM7673292: nexn+/+  biological replicate 2; Danio rerio; RNA Seq,GSM7673292 r1,GSM7673292,1,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP452670,,loader:fastq load.py,NG-33220_nexn_E2_sib3_lib693760_10227_1_1.fastq.gz NG-33220_nexn_E2_sib3_lib693760_10227_1_2.fastq.gz,fastq fastq,9966559304.0,33001852.0,GSM7673292 r1,0:151 1:151,A:2799666848;C:2201108797;G:2237142072;T:2728542087;N:99500,151,151,,,2799666848,2201108797,2237142072,2728542087,99500,SRX21218617,SRS18475796,SRA1684694,"Molecular Cardiology, Internal Medicine II, Uniklinik Ulm","Molecular Cardiology, Internal Medicine II, Uniklinik Ulm",2,0.95433,0.9562,0.11378,0.10948,0.68426,0.68302,0.45622,0.46107,151,151,B,B,biological fallback assumption,illumina,novaseq_era,full_length,random_priming,unknown,bulk,bulk,bulk,,Germany,2023-08-01,Undetermined,Embryo,Whole Organism,All anatomical structures
24655,SRR25487071,SRX21218616,SRS18475795,SRP452670,PRJNA1000968,CRISPR/Cas9 mediated Nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo,GSE239788,Transcriptome Analysis,Nexilin NEXN plays a crucial role in stabilizing the sarcomeric Z disk of striated muscle fibers and  when mutated   leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice  the detailed impact of the  constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated.  We characterized a constitutive homozygous CRISPR/Cas9 mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired  skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably  in contrast to nexn  morphants  CRISPR/Cas9 nexn /  knockout embryos showed a milder phenotype without xxx development of a pronounced  pericardial edema or blood congestion. nexn specific expression analysis as well as whole transcriptome profiling suggest  some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced  and may mediate a sarcomere stabilizing function in nexn /  knockout embryos. Overall design: To investigate the influence of nexn knockout on cardiac and skeletal muslce  we generated a CRISPR/Cas9 mediated nexn  knockout zebrafish model. We then performed gene expression profiling analysis using data obtained from RNA seq.,,pubmed:38114601,,nexn+/+  biological replicate 1,GSM7673291,,source name:whole organism|tissue:whole organism|genotype:WT|geo loc name:missing|collection date:missing,nexn+/+  biological replicate 1,Raw sequencing data is screened for reads originating from rRNA using RiboDetector eurofins genomics  INVIEW transcriptome High quality sequence reads are aligned to the reference genome using STAR Spliced Transcripts Alignment to a Reference  run through Sentieon framework  along with the known gene models. eurofins genomics  INVIEW transcriptome Gene wise quantification is achieved by inspecting transcriptome alignments using RSEM tool. eurofins genomics  INVIEW transcriptome Assembly: GRCz11 Supplementary files format and content: Sample wise  gene wise read counts  TPM value and FPKM value,whole organism,,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,tissue:whole organism|genotype:WT,GSM7673291,GSM7673291: nexn+/+  biological replicate 1; Danio rerio; RNA Seq,GSM7673291 r1,GSM7673291,1,RNA was extracted using the Qiagen RNeasy Mini Kit. 1 25 µg total RNA was used for library preparation. INVIEW transcriptome  done by eurofins Genomics,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP452670,,loader:fastq load.py,NG-33220_nexn_E2_sib2_lib693759_10227_2_1.fastq.gz NG-33220_nexn_E2_sib2_lib693759_10227_2_2.fastq.gz,fastq fastq,13610380470.0,45067485.0,GSM7673291 r1,0:151 1:151,A:3776423931;C:3037607848;G:3100752195;T:3694688167;N:908329,151,151,,,3776423931,3037607848,3100752195,3694688167,908329,SRX21218616,SRS18475795,SRA1684694,"Molecular Cardiology, Internal Medicine II, Uniklinik Ulm","Molecular Cardiology, Internal Medicine II, Uniklinik Ulm",2,0.95714,0.95793,0.10127,0.09788,0.69445,0.69278,0.47084,0.46602,151,151,B,B,biological fallback assumption,illumina,novaseq_era,full_length,random_priming,unknown,bulk,bulk,bulk,,Germany,2023-08-01,Undetermined,Embryo,Whole Organism,All anatomical structures
25182,SRR25670729,SRX21396042,SRS18636200,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA germ ring PAL seq v4,GSM7716871,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA germ ring PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716871,GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER,GSM7716871 r1,GSM7716871,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read2.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep1_raw_read1.fastq.gz,fastq fastq,2834842912.0,9234016.0,GSM7716871 r1,0:52 1:255,A:725811118;C:702410592;G:747583409;T:651018348;N:8019445,52,255,,,725811118,702410592,747583409,651018348,8019445,SRX21396042,SRS18636200,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00023,0.30494,8e-05,0.01793,0.99967,0.99971,0.5,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25183,SRR25670730,SRX21396042,SRS18636200,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA germ ring PAL seq v4,GSM7716871,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA germ ring PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716871,GSM7716871: Fish embryo mRNA germ ring PAL seq v4; Danio rerio; OTHER,GSM7716871 r1,GSM7716871,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_germ_ring_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3473033898.0,11312814.0,GSM7716871 r2,0:52 1:255,A:853178471;C:899976159;G:962100712;T:750811461;N:6967095,52,255,,,853178471,899976159,962100712,750811461,6967095,SRX21396042,SRS18636200,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00049,0.0,0.00012,0.0,0.99941,1.0,0.64864,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25184,SRR25670731,SRX21396041,SRS18636199,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA zfs:0000015 PAL seq v4,GSM7716870,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA zfs:0000015 PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716870,GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER,GSM7716870 r1,GSM7716870,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2695203962.0,8779166.0,GSM7716870 r1,0:52 1:255,A:684535386;C:669852151;G:719008886;T:614209811;N:7597728,52,255,,,684535386,669852151,719008886,614209811,7597728,SRX21396041,SRS18636199,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00117,0.35837,0.00017,0.00682,0.99859,0.99963,0.69473,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25185,SRR25670732,SRX21396041,SRS18636199,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA zfs:0000015 PAL seq v4,GSM7716870,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA zfs:0000015 PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716870,GSM7716870: Fish embryo mRNA zfs:0000015 PAL seq v4; Danio rerio; OTHER,GSM7716870 r1,GSM7716870,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read2.fastq.gz Fish_embryo_mRNA_30percent_epiboly_PAL_seq_v4_rep2_raw_read1.fastq.gz,fastq fastq,3476338446.0,11323578.0,GSM7716870 r2,0:52 1:255,A:841832234;C:898325199;G:968774318;T:760398070;N:7008625,52,255,,,841832234,898325199,968774318,760398070,7008625,SRX21396041,SRS18636199,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00245,0.0,0.00047,0.0,0.99803,1.0,0.58536,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25186,SRR25670733,SRX21396040,SRS18636198,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA sphere PAL seq v4,GSM7716869,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA sphere PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716869,GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER,GSM7716869 r1,GSM7716869,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,3211687254.0,10461522.0,GSM7716869 r1,0:52 1:255,A:813560400;C:775326762;G:854563477;T:759161458;N:9075157,52,255,,,813560400,775326762,854563477,759161458,9075157,SRX21396040,SRS18636198,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00088,0.43191,0.0004,0.01556,0.99916,0.99961,0.55769,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25187,SRR25670734,SRX21396040,SRS18636198,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA sphere PAL seq v4,GSM7716869,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA sphere PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716869,GSM7716869: Fish embryo mRNA sphere PAL seq v4; Danio rerio; OTHER,GSM7716869 r1,GSM7716869,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_sphere_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3179916131.0,10358033.0,GSM7716869 r2,0:52 1:255,A:771755667;C:804256300;G:883166175;T:714242073;N:6495916,52,255,,,771755667,804256300,883166175,714242073,6495916,SRX21396040,SRS18636198,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00186,0.0,0.00088,0.0,0.99862,1.0,0.64705,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25188,SRR25670735,SRX21396039,SRS18636197,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 1024cell PAL seq v4,GSM7716868,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1024cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716868,GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER,GSM7716868 r1,GSM7716868,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2191900794.0,7139742.0,GSM7716868 r1,0:52 1:255,A:571954354;C:551162617;G:572445400;T:490238669;N:6099754,52,255,,,571954354,551162617,572445400,490238669,6099754,SRX21396039,SRS18636197,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.0011,0.43387,6e-05,0.01058,0.99864,0.99971,0.74576,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25189,SRR25670736,SRX21396039,SRS18636197,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 1024cell PAL seq v4,GSM7716868,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1024cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716868,GSM7716868: Fish embryo mRNA 1024cell PAL seq v4; Danio rerio; OTHER,GSM7716868 r1,GSM7716868,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1024cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3840723193.0,12510499.0,GSM7716868 r2,0:52 1:255,A:985340216;C:991311706;G:1034487140;T:821820974;N:7763157,52,255,,,985340216,991311706,1034487140,821820974,7763157,SRX21396039,SRS18636197,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.0021,0.0,0.00026,0.0,0.99859,1.0,0.71022,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25190,SRR25670737,SRX21396038,SRS18636196,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 128cell PAL seq v4,GSM7716867,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 128cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716867,GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER,GSM7716867 r1,GSM7716867,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2104868443.0,6856249.0,GSM7716867 r1,0:52 1:255,A:539122676;C:505234642;G:558793048;T:495876292;N:5841785,52,255,,,539122676,505234642,558793048,495876292,5841785,SRX21396038,SRS18636196,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00035,0.29379,7e-05,0.01129,0.99949,0.99971,0.55882,0.79591,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25191,SRR25670738,SRX21396038,SRS18636196,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 128cell PAL seq v4,GSM7716867,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 128cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716867,GSM7716867: Fish embryo mRNA 128cell PAL seq v4; Danio rerio; OTHER,GSM7716867 r1,GSM7716867,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_128cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3135806371.0,10214353.0,GSM7716867 r2,0:52 1:255,A:776612486;C:774126305;G:853951502;T:724788612;N:6327466,52,255,,,776612486,774126305,853951502,724788612,6327466,SRX21396038,SRS18636196,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00066,0.0,0.00011,0.0,0.99939,1.0,0.55737,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25192,SRR25670739,SRX21396037,SRS18636195,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 8cell PAL seq v4,GSM7716866,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 8cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716866,GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER,GSM7716866 r1,GSM7716866,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2618998887.0,8530941.0,GSM7716866 r1,0:52 1:255,A:673066508;C:641446717;G:706546263;T:590452494;N:7486905,52,255,,,673066508,641446717,706546263,590452494,7486905,SRX21396037,SRS18636195,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.0009,0.43244,0.00014,0.0054,0.99902,0.99967,0.54901,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25193,SRR25670740,SRX21396037,SRS18636195,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 8cell PAL seq v4,GSM7716866,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 8cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716866,GSM7716866: Fish embryo mRNA 8cell PAL seq v4; Danio rerio; OTHER,GSM7716866 r1,GSM7716866,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_8cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3028839589.0,9865927.0,GSM7716866 r2,0:52 1:255,A:756368817;C:758868409;G:836949742;T:670545278;N:6107343,52,255,,,756368817,758868409,836949742,670545278,6107343,SRX21396037,SRS18636195,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00156,0.0,0.00026,0.0,0.99835,1.0,0.44791,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Multi-stage,Embryo,Embryo Imprecise,All anatomical structures
25194,SRR25670741,SRX21396036,SRS18636194,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 1cell PAL seq v4,GSM7716865,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716865,GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER,GSM7716865 r1,GSM7716865,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_1cell_PAL_seq_v4_rep1_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep1_raw_read2.fastq.gz,fastq fastq,2050143851.0,6677993.0,GSM7716865 r1,0:52 1:255,A:536391564;C:527126186;G:557802929;T:422990166;N:5833006,52,255,,,536391564,527126186,557802929,422990166,5833006,SRX21396036,SRS18636194,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00016,0.46479,4e-05,0.01408,0.99979,0.99969,0.54545,1.0,52,255,T,B,mate1 technical by mapping diff,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25195,SRR25670742,SRX21396036,SRS18636194,SRP455680,PRJNA1006406,Control of polyA tail length and translation in vertebrate oocytes and early embryos,GSE241107,Other,During oocyte maturation and early embryonic development  polyA tail lengths strongly influence mRNA translation. However  how tail lengths are controlled at different developmental stages has been unclear. Here  we performed tail length and translational profiling of mRNA reporter libraries each with > 10 million three prime UTR sequence variants in frog oocytes and embryos  and fish embryos. These analyses revealed that the UUUUA motif specifies cytoplasmic polyadenylation and identified diverse context features that modulate the activity of this 5 mer. Additional sequence motifs drive stage specific deadenylation in embryos  and UUUUA and C rich motifs drive tail length independent translational repression in oocytes. A neural network model accurately predicts tail length change during oocyte maturation in frogs  mice  and humans. Analyses of human sequence variants showed that those predicted to disrupt tail length control have been under negative selection  implying that our insights into control of polyA tail length and translation have implications for human health and fertility. Overall design: Sythetic mRNA reporter libraries with random three prime UTR sequences under four different sequence contexts were injected into frog oocytes and embryos and fish embryos. PolyA tail lengths were measured to at different developmental stages to examine sequence motifs that caused tail length changes. At the same time  polyA tail lengths of endogenous mRNAs from frog oocytes and embryos  fish embryos  and mouse oocytes were measured to investiage how their tail lengths were controlled. Please note that sample titles have been updated on Jan 6  2024.,,,,Fish embryo mRNA 1cell PAL seq v4,GSM7716865,,source name:embryo|tissue:embryo|treatment:N1|geo loc name:missing|collection date:missing,Fish embryo mRNA 1cell PAL seq v4,For gene specific tail seq of mRNA reporters  data were processed with a custom script available at https://github.com/coffeebond/MPRA tail seq. For PAL seq v3 or v4  reads were trimmed with cutadapt v3.7 with the parameters “ m 15   quality base=64  q 20 20   match read wildcards  e 0.05  a NNNNATCTCGTATGCCGTCTTCTGCTTG  O 7”. The trimmed reads were mapped using STAR v2.7.1a to the reference database containing the genomic sequences of the organism from which the mRNAs were obtained  the genomic sequences of humans or fish  depending on which spike in RNAs were used  and the sequences of the polyA standards generated previously  with the parameters “  runThreadN 16   runMode alignReads   outFilterMultimapNmax 1   outReadsUnmapped Fastx   outFilterType BySJout   outSAMattributes All   outSAMtype BAM Unsorted SortedByCoordinate”. Uniquely mapped read were furhter processed with a custom script available at https://github.com/coffeebond/PAL seq. Assembly: Homo sapiens: GRCh38.p7 primary assembly. Mus muculus: GRCh38.p4  primary assembly. Xenopus laevis: v10.1 assembly. Danio rerio: GRCz11 assembly. Supplementary files format and content: For gene specific tail seq of mRNA reporters  tab delimited text files with columns indicating 1 sequence of the variable region; 2 median tail length; 3 number of reads; 4–7 tail lengths at quantile 10  25  75  and 90. Supplementary files format and content: For PAL seq v3 or v4  tab delimited files with columns indicating 1 gene ID or polyA site ID; 2 read cluster ID; 3 tail length Library strategy: PAL seq v4,embryo,,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer’s suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,tissue:embryo|treatment:N1,GSM7716865,GSM7716865: Fish embryo mRNA 1cell PAL seq v4; Danio rerio; OTHER,GSM7716865 r1,GSM7716865,1,Frog oocytes and embryos were lysed in ice cold buffer RL 20 mM HEPES pH 7.5  100 mM KCl  5 mM MgCl2  1% [v/v] Triton X 100  100 µg/ml cycloheximide  cOmplete protease inhibitor cocktail [1 tablet per 10 ml buffer]  and 200 units/ml SUPERase•In in a volume of 10 µl per oocyte/embryo by vigorous shaking and pipetting. Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min. The supernatant was transfered to a new tube and mixed with the Tri reagent for RNA isolation. Fish embryos were de chorionated by incubation with 2 mg/ml pronase in E3 medium for 4 min. post removing all E3 medium  Tri Reagent was added to the embryos for RNA isolation. Mouse GV oocytes were collected in 37°C MEM in the presence of milrinone to prevent maturation. Cumulus cells were removed from cumulus oocyte complexes by repeated aspiration through a glass pipette. GV oocytes were then collected in TRI reagent for RNA isolation. Mouse MII oocytes were harvested from the oviducts of hCG induced mice 16 hr  denuded with 3 mg/ml hyaluronidase for 2 min  washed  and then collected in Tri reagent for RNA isolation. For gene specific tail seq of reporter libraries  total RNA with reporter mRNA libraries was ligated to a pre adenylated 3ʹ adapter directly in most cases  but for the N60 library injected into fish embryos and frog oocytes  the N37 PAS N17 library injected into fish embryos and frog oocytes  and the CPEmos N60 and N60LC PASmos libraries injected into frog oocytes  reporter library mRNAs were enriched by anti sense oligo capture with biotinylated oligos. RNA isolated from the oocyte or embryo lysate was mixed with 8 pmol KXSH009  8 pmol KXSH010  and 2x SSC 0.3 M NaCl  30 mM sodium citrate pH 7.0 in a total of 50 µl. The RNA and oligos were annealed by incubation at 70°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 40 µl MyOne Streptavidin C1 beads Thermo Fisher  65002 and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack and removed. The beads were washed twice with 300 µl 1xB&W buffer 5 mM Tris HCl pH 7.5  0.5 mM EDTA  1 M NaCl and once with 300 µl 2x SSC. The RNA was eluted from the beads first with 100 µl 10 mM HEPES pH 7.5 at 65°C for 3 min and then second with 100 µl water at 65°C for 3 min. The eluates were combined  precipitated with ethanol  and resuspended in 6.5 µl water. The anti sense oligo enriched RNA or the RNA isolated from oocyte or embryo lysates was ligated to a pre adenylated 3ʹ adapter in a 10 µl reaction containing 5 µM 3ʹ adapter KXS330  50 mM HEPES pH 7.5  10 mM MgCl2  10 mM dithiothreitol  1 unit/µl T4 RNA ligase 1 New England Biolabs  M0204S. The ligation reaction was incubated at 23°C for 150 min. post ligation  RNA was extracted with phenol/chloroform  precipitated with ethanol  and resuspended in 11.4 µl water. The ligated RNA was mixed with 0.6 µl 100 µM reverse transcription primer KXS037 in a total volume of 12 µl  incubated at 65°C for 5 min  and cooled on ice for 1 min. The annealed RNA was reverse transcribed in a 20 µl reaction containing 1x First Strand Buffer  500 µM dNTPs  5 mM dithiothreitol  1 unit/µl SUPERase•In  and 200 units SuperScript III Thermo Fisher  18080044 at 50°C for 1 hr. post reverse transcription  RNA was hydrolyzed with 3.3 µl 1 M NaOH at 90°C for 10 min  followed by neutralization with 36.7 µl 1 M HEPES pH 7.5 and the cDNA was collected by desalting with a Micro Bio Spin P 30 column. The cDNA library was amplified in a 50 µl PCR reaction with KXS037 and a barcoded primer Supplemental using the KAPA HiFi HotStart Kits following the manufacturer's suggested protocol for 10–15 cycles. The PCR amplified library was cleaned up twice with AMPure XP beads Beckman Coulter  A63881 with a beads to sample ratio of 1.2. For sequencing of endogenous mRNA polyA tail length  libraries were prepared with PAL seq v3 for frog oocytes or PAL seq v4 for frog embryos  fish embryos  and mouse oocytes as described previously PMID: 34213414. When preparing the sequencing libraries of mRNAs from fish embryos  a different 3ʹ adapter KXS013 was used for 3ʹ end ligation  and polyA selected mRNA from HeLa cells was used as spike in  replacing polyA selected mRNA from zebrafish ZF4 cell line. The first round of sequencing results suggested that a large fraction of the fish mRNA libraries was 5.8S rRNA. The cDNA of 5.8S rRNA was depleted from the cDNA libraries with an antisense oligo. The seven cDNA libraries made from mRNAs of zebrafish embryos at different stages were mixed at roughly equal molar ratios in a total of 5 fmol. Fifty pmol KXSH015 and 2x SSC were added in a total of 100 µl. The cDNAs and the oligo were annealed by incubation at 65°C for 5 min and then slowly cooling to 23°C at 0.1°C/sec. The annealed mixture was combined with 100 µl MyOne Streptavidin C1 beads and incubated for 20 min at 23°C on a thermal mixer  shaking with 15 sec on and 1 min 45 sec off. The supernatant was separated from the beads with a magnetic rack. The beads were washed once with 200 µl 1xB&W buffer. The supernatant and the wash were combined  precipitated with ethanol  and resuspended in 30 µl water. The oligo depleted libraries were sequenced again as a technical replicate. Sequencing data of replicates were merged for each sample post monitoring consistency.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP455680,,,Fish_embryo_mRNA_1cell_PAL_seq_v4_rep2_raw_read1.fastq.gz Fish_embryo_mRNA_1cell_PAL_seq_v4_rep2_raw_read2.fastq.gz,fastq fastq,3406093776.0,11094768.0,GSM7716865 r2,0:52 1:255,A:868054279;C:890446070;G:945289336;T:695324057;N:6980034,52,255,,,868054279,890446070,945289336,695324057,6980034,SRX21396036,SRS18636194,SRA1694849,Whitehead Institute,Whitehead Institute,2,0.00049,0.0,0.00014,0.0,0.99939,1.0,0.58974,,52,255,T,T,mates < 9% mapping rate,illumina,hiseq_era,unknown,poly_a,unknown,bulk,bulk,bulk,,United States,2023-08-17,Zygote,Embryo,Embryo Imprecise,All anatomical structures
25273,SRR25764091,SRX21486763,SRS18719063,SRP457108,PRJNA1009807,Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [RNA Seq],GSE241752,Transcriptome Analysis,Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points  namelyat the 256 cell 2.5 hpf  1000 cell 3 hpf  sphere 4 hpf  shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis  polysome profiling  ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.,parent bioproject:PRJNA1009800,pubmed:39402326,,WT bud 10 hpf RNA seq rep2,GSM7734768,,source name:Gastrula|strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|genotype:WT|geo loc name:missing|collection date:missing,WT bud 10 hpf RNA seq rep2,3’ adapters were trimmed using Trim Galore v0.6.4 with default settings  retaining reads of length ≥20. Reads were aligned to the GRCz11 zebrafish genome using STAR v2.6.1c with the following parameters:   outSAMtype BAM SortedByCoordinate   outFilterMultimapNmax 1   outFilterMismatchNmax 1   quantMode TranscriptomeSAM GeneCounts   alignEndsType Local   seedSearchStartLmax 14   alignIntronMax 10000   outFilterIntronMotifs RemoveNoncanonicalUnannotated. featureCounts v1.6.2 was used to count reads overlapping a filtered set of protein coding gene annotations from the GENCODE basic gene annotation. Differential gene expression analysis was performed using DESEq2 v1.38.1 with default settings and gene counts from featureCounts. Assembly: GRCz11 Supplementary files format and content: csv; transcripts per million TPM counts per transcript in MANE annotation,Gastrula,unperturbed growth conditions in E3 medium for zebrafish embryos.,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle.,strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|genotype:WT,GSM7734768,GSM7734768: WT bud 10 hpf RNA seq rep2; Danio rerio; RNA Seq,GSM7734768 r1,GSM7734768,1,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP457108,,,WT_mRNA_bud_2.fastq.gz,fastq,2470912004.0,31168086.0,GSM7734768 r1,0:79.28,A:656817108;C:568241258;G:504262095;T:741507737;N:83806,79,,,,656817108,568241258,504262095,741507737,83806,SRX21486763,SRS18719063,SRA1700431,"Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry",Max Planck Institute of Biochemistry,1,0.91479,,0.27298,,0.74231,,0.57302,,79,,B,,usable mapping rate,illumina,nextseq,unknown,small_rna,unknown,bulk,bulk,bulk,,Germany,2023-08-28,Gastrula,Embryo,Whole Organism,All anatomical structures
25274,SRR25764092,SRX21486762,SRS18719064,SRP457108,PRJNA1009807,Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [RNA Seq],GSE241752,Transcriptome Analysis,Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points  namelyat the 256 cell 2.5 hpf  1000 cell 3 hpf  sphere 4 hpf  shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis  polysome profiling  ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.,parent bioproject:PRJNA1009800,pubmed:39402326,,WT bud 10 hpf RNA seq rep1,GSM7734767,,source name:Gastrula|strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|genotype:WT|geo loc name:missing|collection date:missing,WT bud 10 hpf RNA seq rep1,3’ adapters were trimmed using Trim Galore v0.6.4 with default settings  retaining reads of length ≥20. Reads were aligned to the GRCz11 zebrafish genome using STAR v2.6.1c with the following parameters:   outSAMtype BAM SortedByCoordinate   outFilterMultimapNmax 1   outFilterMismatchNmax 1   quantMode TranscriptomeSAM GeneCounts   alignEndsType Local   seedSearchStartLmax 14   alignIntronMax 10000   outFilterIntronMotifs RemoveNoncanonicalUnannotated. featureCounts v1.6.2 was used to count reads overlapping a filtered set of protein coding gene annotations from the GENCODE basic gene annotation. Differential gene expression analysis was performed using DESEq2 v1.38.1 with default settings and gene counts from featureCounts. Assembly: GRCz11 Supplementary files format and content: csv; transcripts per million TPM counts per transcript in MANE annotation,Gastrula,unperturbed growth conditions in E3 medium for zebrafish embryos.,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle.,strain:TLAB strain|tissue:Gastrula|developmental stage:Bud 10 hpf|genotype:WT,GSM7734767,GSM7734767: WT bud 10 hpf RNA seq rep1; Danio rerio; RNA Seq,GSM7734767 r1,GSM7734767,1,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP457108,,,WT_mRNA_bud_1.fastq.gz,fastq,2153494756.0,27140032.0,GSM7734767 r1,0:79.35,A:560793143;C:508962676;G:445126772;T:638539310;N:72855,79,,,,560793143,508962676,445126772,638539310,72855,SRX21486762,SRS18719064,SRA1700431,"Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry",Max Planck Institute of Biochemistry,1,0.88728,,0.25493,,0.74369,,0.56589,,80,,B,,usable mapping rate,illumina,nextseq,unknown,small_rna,unknown,bulk,bulk,bulk,,Germany,2023-08-28,Gastrula,Embryo,Whole Organism,All anatomical structures
25275,SRR25764093,SRX21486761,SRS18719061,SRP457108,PRJNA1009807,Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [RNA Seq],GSE241752,Transcriptome Analysis,Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points  namelyat the 256 cell 2.5 hpf  1000 cell 3 hpf  sphere 4 hpf  shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis  polysome profiling  ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.,parent bioproject:PRJNA1009800,pubmed:39402326,,WT sphere 4 hpf RNA seq rep2,GSM7734766,,source name:Blastula|strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|genotype:WT|geo loc name:missing|collection date:missing,WT sphere 4 hpf RNA seq rep2,3’ adapters were trimmed using Trim Galore v0.6.4 with default settings  retaining reads of length ≥20. Reads were aligned to the GRCz11 zebrafish genome using STAR v2.6.1c with the following parameters:   outSAMtype BAM SortedByCoordinate   outFilterMultimapNmax 1   outFilterMismatchNmax 1   quantMode TranscriptomeSAM GeneCounts   alignEndsType Local   seedSearchStartLmax 14   alignIntronMax 10000   outFilterIntronMotifs RemoveNoncanonicalUnannotated. featureCounts v1.6.2 was used to count reads overlapping a filtered set of protein coding gene annotations from the GENCODE basic gene annotation. Differential gene expression analysis was performed using DESEq2 v1.38.1 with default settings and gene counts from featureCounts. Assembly: GRCz11 Supplementary files format and content: csv; transcripts per million TPM counts per transcript in MANE annotation,Blastula,unperturbed growth conditions in E3 medium for zebrafish embryos.,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle.,strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|genotype:WT,GSM7734766,GSM7734766: WT sphere 4 hpf RNA seq rep2; Danio rerio; RNA Seq,GSM7734766 r1,GSM7734766,1,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP457108,,,WT_mRNA_sphere_2.fastq.gz,fastq,2455317157.0,30934591.0,GSM7734766 r1,0:79.37,A:614860604;C:588586084;G:522132289;T:729655621;N:82559,79,,,,614860604,588586084,522132289,729655621,82559,SRX21486761,SRS18719061,SRA1700431,"Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry",Max Planck Institute of Biochemistry,1,0.94124,,0.10502,,0.74876,,0.57752,,80,,B,,usable mapping rate,illumina,nextseq,unknown,small_rna,unknown,bulk,bulk,bulk,,Germany,2023-08-28,Blastula,Embryo,Whole Organism,All anatomical structures
25276,SRR25764094,SRX21486760,SRS18719060,SRP457108,PRJNA1009807,Dynamics of the zebrafish tRNAome during the maternal to zygotic transition [RNA Seq],GSE241752,Transcriptome Analysis,Time course analysis of tRNA abundance during zebrafish early embryonic development. Overall design: Wild type TLAB strain zebrafish embryos were grown in standard housing conditions.Unfertilized eggs 0 hpf were collected or embryos were staged and collected at consecutive developmental time points  namelyat the 256 cell 2.5 hpf  1000 cell 3 hpf  sphere 4 hpf  shield 6 hpf and bud 10 hpf stages. Eggs and embryos were either flash frozen in liquid nitrogen or immediately processed. Samples were used for western blotting analysis  polysome profiling  ribosome profiling or mRNA and tRNA sequencing for investigating the regulation of tRNA gene expression and translational status during early zebrafish embryogenesis.,parent bioproject:PRJNA1009800,pubmed:39402326,,WT sphere 4 hpf RNA seq rep1,GSM7734765,,source name:Blastula|strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|genotype:WT|geo loc name:missing|collection date:missing,WT sphere 4 hpf RNA seq rep1,3’ adapters were trimmed using Trim Galore v0.6.4 with default settings  retaining reads of length ≥20. Reads were aligned to the GRCz11 zebrafish genome using STAR v2.6.1c with the following parameters:   outSAMtype BAM SortedByCoordinate   outFilterMultimapNmax 1   outFilterMismatchNmax 1   quantMode TranscriptomeSAM GeneCounts   alignEndsType Local   seedSearchStartLmax 14   alignIntronMax 10000   outFilterIntronMotifs RemoveNoncanonicalUnannotated. featureCounts v1.6.2 was used to count reads overlapping a filtered set of protein coding gene annotations from the GENCODE basic gene annotation. Differential gene expression analysis was performed using DESEq2 v1.38.1 with default settings and gene counts from featureCounts. Assembly: GRCz11 Supplementary files format and content: csv; transcripts per million TPM counts per transcript in MANE annotation,Blastula,unperturbed growth conditions in E3 medium for zebrafish embryos.,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,Embryos were grown in standard housing conditions namely28°C at a 14/10 hour light/dark cycle.,strain:TLAB strain|tissue:Blastula|developmental stage:Sphere 4 hpf|genotype:WT,GSM7734765,GSM7734765: WT sphere 4 hpf RNA seq rep1; Danio rerio; RNA Seq,GSM7734765 r1,GSM7734765,1,50 whole embryos were collected per sample and immediately lysed in 800uL LiDS/LET buffer5% Lithium dodecyl sulfate in 20 mM Tris HCl pH=7.4  100 mM LiCl  2 mM EDTA  5 mM DTT  pH 7.4. 250 ng of the same total RNA used for mim tRNAseq library preparation were used for mRNA Seq library construction with the Zymo Seq RiboFree Total RNA Library Kit Zymo Research  #R3000. Libraries were sequenced on a NextSeq 500 platform.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP457108,,,WT_mRNA_sphere_1.fastq.gz,fastq,2399640874.0,30232118.0,GSM7734765 r1,0:79.37,A:589069860;C:593745131;G:511439690;T:705305674;N:80519,79,,,,589069860,593745131,511439690,705305674,80519,SRX21486760,SRS18719060,SRA1700431,"Mechanisms of Protein Biogenesis, Max Planck Institute for Biochemistry",Max Planck Institute of Biochemistry,1,0.85882,,0.11848,,0.75122,,0.5883,,80,,B,,usable mapping rate,illumina,nextseq,unknown,small_rna,unknown,bulk,bulk,bulk,,Germany,2023-08-28,Blastula,Embryo,Whole Organism,All anatomical structures
28113,SRR26209648,SRX21920662,SRS19005181,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 3,GSM7812991,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812991,GSM7812991: unDMSO t1 WT 3; Danio rerio; RNA Seq,GSM7812991 r1,GSM7812991,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A30_S48_L005_R1_001.fastq.gz,fastq,616449359.0,6103459.0,GSM7812991 r1,0:101,A:152808626;C:149738763;G:141639387;T:172257280;N:5303,101,,,,152808626,149738763,141639387,172257280,5303,SRX21920662,SRS19005181,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94246,,0.08766,,0.69443,,0.4842,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28114,SRR26209649,SRX21920662,SRS19005181,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 3,GSM7812991,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812991,GSM7812991: unDMSO t1 WT 3; Danio rerio; RNA Seq,GSM7812991 r1,GSM7812991,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A30_S48_L006_R1_001.fastq.gz,fastq,609999297.0,6039597.0,GSM7812991 r2,0:101,A:151226657;C:148143193;G:140088806;T:170530812;N:9829,101,,,,151226657,148143193,140088806,170530812,9829,SRX21920662,SRS19005181,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94201,,0.08569,,0.69572,,0.48692,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28115,SRR26209650,SRX21920662,SRS19005181,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 3,GSM7812991,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812991,GSM7812991: unDMSO t1 WT 3; Danio rerio; RNA Seq,GSM7812991 r1,GSM7812991,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A30_S48_L007_R1_001.fastq.gz,fastq,543730470.0,5383470.0,GSM7812991 r3,0:101,A:134685766;C:132190291;G:124881864;T:151967639;N:4910,101,,,,134685766,132190291,124881864,151967639,4910,SRX21920662,SRS19005181,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.9431,,0.08733,,0.69524,,0.48528,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28116,SRR26209651,SRX21920662,SRS19005181,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 3,GSM7812991,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812991,GSM7812991: unDMSO t1 WT 3; Danio rerio; RNA Seq,GSM7812991 r1,GSM7812991,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A30_S16_L007_R1_001.fastq.gz,fastq,649354149.0,6429249.0,GSM7812991 r4,0:101,A:160756303;C:158145998;G:149623680;T:180816390;N:11778,101,,,,160756303,158145998,149623680,180816390,11778,SRX21920662,SRS19005181,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94438,,0.08618,,0.69473,,0.48809,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28117,SRR26209752,SRX21920662,SRS19005181,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 3,GSM7812991,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812991,GSM7812991: unDMSO t1 WT 3; Danio rerio; RNA Seq,GSM7812991 r1,GSM7812991,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A30_S16_L008_R1_001.fastq.gz,fastq,650450504.0,6440104.0,GSM7812991 r5,0:101,A:161054597;C:158340761;G:149784923;T:181251361;N:18862,101,,,,161054597,158340761,149784923,181251361,18862,SRX21920662,SRS19005181,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94338,,0.08713,,0.6968,,0.48443,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28118,SRR26209652,SRX21920661,SRS19005180,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 2,GSM7812990,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812990,GSM7812990: unDMSO t1 WT 2; Danio rerio; RNA Seq,GSM7812990 r1,GSM7812990,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A29_S46_L005_R1_001.fastq.gz,fastq,593068162.0,5871962.0,GSM7812990 r1,0:101,A:147906639;C:143944406;G:137307101;T:163904929;N:5087,101,,,,147906639,143944406,137307101,163904929,5087,SRX21920661,SRS19005180,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94371,,0.06473,,0.69378,,0.47765,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28119,SRR26209653,SRX21920661,SRS19005180,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 2,GSM7812990,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812990,GSM7812990: unDMSO t1 WT 2; Danio rerio; RNA Seq,GSM7812990 r1,GSM7812990,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A29_S46_L006_R1_001.fastq.gz,fastq,588323081.0,5824981.0,GSM7812990 r2,0:101,A:146630863;C:142829925;G:136124185;T:162728186;N:9922,101,,,,146630863,142829925,136124185,162728186,9922,SRX21920661,SRS19005180,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94282,,0.06357,,0.69418,,0.47665,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28120,SRR26209654,SRX21920661,SRS19005180,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 2,GSM7812990,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812990,GSM7812990: unDMSO t1 WT 2; Danio rerio; RNA Seq,GSM7812990 r1,GSM7812990,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A29_S46_L007_R1_001.fastq.gz,fastq,521265747.0,5161047.0,GSM7812990 r3,0:101,A:129905878;C:126622019;G:120526207;T:144206853;N:4790,101,,,,129905878,126622019,120526207,144206853,4790,SRX21920661,SRS19005180,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94508,,0.06499,,0.69367,,0.4834,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28121,SRR26209655,SRX21920661,SRS19005180,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 2,GSM7812990,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812990,GSM7812990: unDMSO t1 WT 2; Danio rerio; RNA Seq,GSM7812990 r1,GSM7812990,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A29_S26_L007_R1_001.fastq.gz,fastq,623834580.0,6176580.0,GSM7812990 r4,0:101,A:155642981;C:151651242;G:144681951;T:171847192;N:11214,101,,,,155642981,151651242,144681951,171847192,11214,SRX21920661,SRS19005180,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94531,,0.06514,,0.69225,,0.48187,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28122,SRR26209656,SRX21920661,SRS19005180,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 2,GSM7812990,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812990,GSM7812990: unDMSO t1 WT 2; Danio rerio; RNA Seq,GSM7812990 r1,GSM7812990,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A29_S26_L008_R1_001.fastq.gz,fastq,622155960.0,6159960.0,GSM7812990 r5,0:101,A:155145221;C:151231599;G:144290581;T:171470641;N:17918,101,,,,155145221,151231599,144290581,171470641,17918,SRX21920661,SRS19005180,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94319,,0.06413,,0.69449,,0.48159,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28123,SRR26209657,SRX21920660,SRS19005179,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 1,GSM7812989,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812989,GSM7812989: unDMSO t1 WT 1; Danio rerio; RNA Seq,GSM7812989 r1,GSM7812989,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A28_S45_L005_R1_001.fastq.gz,fastq,602520550.0,5965550.0,GSM7812989 r1,0:101,A:149619230;C:146559346;G:139674646;T:166662143;N:5185,101,,,,149619230,146559346,139674646,166662143,5185,SRX21920660,SRS19005179,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94449,,0.06296,,0.69487,,0.48,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28124,SRR26209658,SRX21920660,SRS19005179,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 1,GSM7812989,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812989,GSM7812989: unDMSO t1 WT 1; Danio rerio; RNA Seq,GSM7812989 r1,GSM7812989,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A28_S45_L006_R1_001.fastq.gz,fastq,596942522.0,5910322.0,GSM7812989 r2,0:101,A:148193962;C:145221539;G:138267073;T:165249833;N:10115,101,,,,148193962,145221539,138267073,165249833,10115,SRX21920660,SRS19005179,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.9446,,0.06327,,0.69562,,0.48183,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28125,SRR26209659,SRX21920660,SRS19005179,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 1,GSM7812989,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812989,GSM7812989: unDMSO t1 WT 1; Danio rerio; RNA Seq,GSM7812989 r1,GSM7812989,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A28_S45_L007_R1_001.fastq.gz,fastq,532539064.0,5272664.0,GSM7812989 r3,0:101,A:132217323;C:129660296;G:123303893;T:147352732;N:4820,101,,,,132217323,129660296,123303893,147352732,4820,SRX21920660,SRS19005179,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94579,,0.06237,,0.69554,,0.46658,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28126,SRR26209660,SRX21920660,SRS19005179,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 1,GSM7812989,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812989,GSM7812989: unDMSO t1 WT 1; Danio rerio; RNA Seq,GSM7812989 r1,GSM7812989,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A28_S29_L007_R1_001.fastq.gz,fastq,633337569.0,6270669.0,GSM7812989 r4,0:101,A:157286526;C:154336036;G:147137950;T:174565295;N:11762,101,,,,157286526,154336036,147137950,174565295,11762,SRX21920660,SRS19005179,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94562,,0.06294,,0.69489,,0.47406,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28127,SRR26209661,SRX21920660,SRS19005179,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 WT 1,GSM7812989,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 WT 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:WT|treatment:unDMSO,GSM7812989,GSM7812989: unDMSO t1 WT 1; Danio rerio; RNA Seq,GSM7812989 r1,GSM7812989,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A28_S29_L008_R1_001.fastq.gz,fastq,636051540.0,6297540.0,GSM7812989 r5,0:101,A:157845668;C:154972063;G:147765532;T:175449801;N:18476,101,,,,157845668,154972063,147765532,175449801,18476,SRX21920660,SRS19005179,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.9471,,0.06288,,0.69536,,0.4797,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28128,SRR26209662,SRX21920659,SRS19005178,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 3,GSM7812988,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812988,GSM7812988: unDMSO t1 K2 3; Danio rerio; RNA Seq,GSM7812988 r1,GSM7812988,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A36_S54_L005_R1_001.fastq.gz,fastq,674188635.0,6675135.0,GSM7812988 r1,0:101,A:173104307;C:161481671;G:152542615;T:187053812;N:6230,101,,,,173104307,161481671,152542615,187053812,6230,SRX21920659,SRS19005178,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94039,,0.07795,,0.69877,,0.48386,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28129,SRR26209663,SRX21920659,SRS19005178,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 3,GSM7812988,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812988,GSM7812988: unDMSO t1 K2 3; Danio rerio; RNA Seq,GSM7812988 r1,GSM7812988,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A36_S54_L006_R1_001.fastq.gz,fastq,667048339.0,6604439.0,GSM7812988 r2,0:101,A:171316049;C:159786547;G:150743446;T:185190228;N:12069,101,,,,171316049,159786547,150743446,185190228,12069,SRX21920659,SRS19005178,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94064,,0.07881,,0.69767,,0.48162,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28130,SRR26209664,SRX21920659,SRS19005178,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 3,GSM7812988,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812988,GSM7812988: unDMSO t1 K2 3; Danio rerio; RNA Seq,GSM7812988 r1,GSM7812988,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A36_S54_L007_R1_001.fastq.gz,fastq,596645683.0,5907383.0,GSM7812988 r3,0:101,A:153131508;C:143126741;G:134745614;T:165635953;N:5867,101,,,,153131508,143126741,134745614,165635953,5867,SRX21920659,SRS19005178,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94156,,0.07795,,0.69751,,0.4833,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28131,SRR26209665,SRX21920659,SRS19005178,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 3,GSM7812988,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812988,GSM7812988: unDMSO t1 K2 3; Danio rerio; RNA Seq,GSM7812988 r1,GSM7812988,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A36_S12_L007_R1_001.fastq.gz,fastq,712385926.0,7053326.0,GSM7812988 r4,0:101,A:183100436;C:171026605;G:161510527;T:196734608;N:13750,101,,,,183100436,171026605,161510527,196734608,13750,SRX21920659,SRS19005178,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94298,,0.07892,,0.6997,,0.48331,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28132,SRR26209666,SRX21920659,SRS19005178,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 3,GSM7812988,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812988,GSM7812988: unDMSO t1 K2 3; Danio rerio; RNA Seq,GSM7812988 r1,GSM7812988,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A36_S12_L008_R1_001.fastq.gz,fastq,714661355.0,7075855.0,GSM7812988 r5,0:101,A:183454698;C:171554090;G:162064705;T:197567065;N:20797,101,,,,183454698,171554090,162064705,197567065,20797,SRX21920659,SRS19005178,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94316,,0.07877,,0.70051,,0.48354,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28133,SRR26209667,SRX21920658,SRS19005177,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 2,GSM7812987,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812987,GSM7812987: unDMSO t1 K2 2; Danio rerio; RNA Seq,GSM7812987 r1,GSM7812987,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A35_S53_L005_R1_001.fastq.gz,fastq,611900723.0,6058423.0,GSM7812987 r1,0:101,A:154668392;C:147817141;G:139653400;T:169756342;N:5448,101,,,,154668392,147817141,139653400,169756342,5448,SRX21920658,SRS19005177,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94636,,0.08022,,0.69962,,0.48201,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28134,SRR26209668,SRX21920658,SRS19005177,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 2,GSM7812987,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812987,GSM7812987: unDMSO t1 K2 2; Danio rerio; RNA Seq,GSM7812987 r1,GSM7812987,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A35_S53_L006_R1_001.fastq.gz,fastq,602971818.0,5970018.0,GSM7812987 r2,0:101,A:152346877;C:145640105;G:137576284;T:167398281;N:10271,101,,,,152346877,145640105,137576284,167398281,10271,SRX21920658,SRS19005177,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94549,,0.08086,,0.69781,,0.47842,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28135,SRR26209669,SRX21920658,SRS19005177,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 2,GSM7812987,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812987,GSM7812987: unDMSO t1 K2 2; Danio rerio; RNA Seq,GSM7812987 r1,GSM7812987,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A35_S53_L007_R1_001.fastq.gz,fastq,539246575.0,5339075.0,GSM7812987 r3,0:101,A:136317506;C:130390122;G:122927887;T:149606028;N:5032,101,,,,136317506,130390122,122927887,149606028,5032,SRX21920658,SRS19005177,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94653,,0.08075,,0.69704,,0.48374,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28136,SRR26209670,SRX21920658,SRS19005177,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 2,GSM7812987,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812987,GSM7812987: unDMSO t1 K2 2; Danio rerio; RNA Seq,GSM7812987 r1,GSM7812987,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A35_S47_L007_R1_001.fastq.gz,fastq,643197593.0,6368293.0,GSM7812987 r4,0:101,A:162743675;C:155711536;G:147135700;T:177594459;N:12223,101,,,,162743675,155711536,147135700,177594459,12223,SRX21920658,SRS19005177,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94757,,0.08023,,0.70055,,0.48458,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28137,SRR26209671,SRX21920658,SRS19005177,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 2,GSM7812987,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812987,GSM7812987: unDMSO t1 K2 2; Danio rerio; RNA Seq,GSM7812987 r1,GSM7812987,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A35_S47_L008_R1_001.fastq.gz,fastq,644356063.0,6379763.0,GSM7812987 r5,0:101,A:162915651;C:155935181;G:147406641;T:178079118;N:19472,101,,,,162915651,155935181,147406641,178079118,19472,SRX21920658,SRS19005177,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94817,,0.08218,,0.70017,,0.48286,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28138,SRR26209672,SRX21920657,SRS19005176,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 1,GSM7812986,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812986,GSM7812986: unDMSO t1 K2 1; Danio rerio; RNA Seq,GSM7812986 r1,GSM7812986,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A34_S52_L005_R1_001.fastq.gz,fastq,615996374.0,6098974.0,GSM7812986 r1,0:101,A:156934542;C:147417998;G:139301915;T:172336421;N:5498,101,,,,156934542,147417998,139301915,172336421,5498,SRX21920657,SRS19005176,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94179,,0.07628,,0.69512,,0.48041,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28139,SRR26209673,SRX21920657,SRS19005176,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 1,GSM7812986,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812986,GSM7812986: unDMSO t1 K2 1; Danio rerio; RNA Seq,GSM7812986 r1,GSM7812986,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A34_S52_L006_R1_001.fastq.gz,fastq,608875571.0,6028471.0,GSM7812986 r2,0:101,A:155127967;C:145701864;G:137575885;T:170459017;N:10838,101,,,,155127967,145701864,137575885,170459017,10838,SRX21920657,SRS19005176,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94248,,0.07563,,0.69512,,0.47982,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28140,SRR26209674,SRX21920657,SRS19005176,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 1,GSM7812986,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812986,GSM7812986: unDMSO t1 K2 1; Danio rerio; RNA Seq,GSM7812986 r1,GSM7812986,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A34_S52_L007_R1_001.fastq.gz,fastq,539739455.0,5343955.0,GSM7812986 r3,0:101,A:137431217;C:129328124;G:121891472;T:151083373;N:5269,101,,,,137431217,129328124,121891472,151083373,5269,SRX21920657,SRS19005176,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.9438,,0.07511,,0.69654,,0.48083,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28141,SRR26209675,SRX21920657,SRS19005176,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 1,GSM7812986,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812986,GSM7812986: unDMSO t1 K2 1; Danio rerio; RNA Seq,GSM7812986 r1,GSM7812986,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A34_S65_L007_R1_001.fastq.gz,fastq,648984489.0,6425589.0,GSM7812986 r4,0:101,A:165552674;C:155580501;G:147078092;T:180761105;N:12117,101,,,,165552674,155580501,147078092,180761105,12117,SRX21920657,SRS19005176,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94513,,0.07694,,0.69451,,0.48174,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28142,SRR26209676,SRX21920657,SRS19005176,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K2 1,GSM7812986,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K2 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K2|treatment:unDMSO,GSM7812986,GSM7812986: unDMSO t1 K2 1; Danio rerio; RNA Seq,GSM7812986 r1,GSM7812986,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A34_S65_L008_R1_001.fastq.gz,fastq,651988330.0,6455330.0,GSM7812986 r5,0:101,A:166130853;C:156282200;G:147738788;T:181817695;N:18794,101,,,,166130853,156282200,147738788,181817695,18794,SRX21920657,SRS19005176,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94437,,0.0759,,0.69597,,0.48083,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28143,SRR26209677,SRX21920656,SRS19005175,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 3,GSM7812985,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812985,GSM7812985: unDMSO t1 K1 3; Danio rerio; RNA Seq,GSM7812985 r1,GSM7812985,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A33_S51_L005_R1_001.fastq.gz,fastq,621669544.0,6155144.0,GSM7812985 r1,0:101,A:157346917;C:148083763;G:141122336;T:175110946;N:5582,101,,,,157346917,148083763,141122336,175110946,5582,SRX21920656,SRS19005175,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94207,,0.06859,,0.69473,,0.48838,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28144,SRR26209678,SRX21920656,SRS19005175,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 3,GSM7812985,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812985,GSM7812985: unDMSO t1 K1 3; Danio rerio; RNA Seq,GSM7812985 r1,GSM7812985,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A33_S51_L006_R1_001.fastq.gz,fastq,613437842.0,6073642.0,GSM7812985 r2,0:101,A:155200714;C:146135271;G:139223090;T:172868018;N:10749,101,,,,155200714,146135271,139223090,172868018,10749,SRX21920656,SRS19005175,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94144,,0.06885,,0.69471,,0.48996,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28145,SRR26209679,SRX21920656,SRS19005175,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 3,GSM7812985,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812985,GSM7812985: unDMSO t1 K1 3; Danio rerio; RNA Seq,GSM7812985 r1,GSM7812985,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A33_S51_L007_R1_001.fastq.gz,fastq,546684518.0,5412718.0,GSM7812985 r3,0:101,A:138292090;C:130364936;G:123978492;T:154043685;N:5315,101,,,,138292090,130364936,123978492,154043685,5315,SRX21920656,SRS19005175,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94375,,0.06988,,0.69589,,0.48741,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28146,SRR26209680,SRX21920656,SRS19005175,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 3,GSM7812985,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812985,GSM7812985: unDMSO t1 K1 3; Danio rerio; RNA Seq,GSM7812985 r1,GSM7812985,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A33_S43_L007_R1_001.fastq.gz,fastq,651065089.0,6446189.0,GSM7812985 r4,0:101,A:164874330;C:155389435;G:148130531;T:182658473;N:12320,101,,,,164874330,155389435,148130531,182658473,12320,SRX21920656,SRS19005175,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94247,,0.06954,,0.69538,,0.48283,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28147,SRR26209681,SRX21920656,SRS19005175,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 3,GSM7812985,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 3,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812985,GSM7812985: unDMSO t1 K1 3; Danio rerio; RNA Seq,GSM7812985 r1,GSM7812985,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A33_S43_L008_R1_001.fastq.gz,fastq,652838952.0,6463752.0,GSM7812985 r5,0:101,A:165235507;C:155756581;G:148483290;T:183344035;N:19539,101,,,,165235507,155756581,148483290,183344035,19539,SRX21920656,SRS19005175,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94297,,0.06917,,0.69593,,0.4827,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28148,SRR26209682,SRX21920655,SRS19005174,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 2,GSM7812984,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812984,GSM7812984: unDMSO t1 K1 2; Danio rerio; RNA Seq,GSM7812984 r1,GSM7812984,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A32_S50_L005_R1_001.fastq.gz,fastq,531938215.0,5266715.0,GSM7812984 r1,0:101,A:131083905;C:128715617;G:122601298;T:149532970;N:4425,101,,,,131083905,128715617,122601298,149532970,4425,SRX21920655,SRS19005174,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94249,,0.07108,,0.69485,,0.47386,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28149,SRR26209683,SRX21920655,SRS19005174,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 2,GSM7812984,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812984,GSM7812984: unDMSO t1 K1 2; Danio rerio; RNA Seq,GSM7812984 r1,GSM7812984,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A32_S50_L006_R1_001.fastq.gz,fastq,527781863.0,5225563.0,GSM7812984 r2,0:101,A:130119726;C:127646784;G:121549831;T:148456993;N:8529,101,,,,130119726,127646784,121549831,148456993,8529,SRX21920655,SRS19005174,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94199,,0.0713,,0.69337,,0.473,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28150,SRR26209684,SRX21920655,SRS19005174,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 2,GSM7812984,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812984,GSM7812984: unDMSO t1 K1 2; Danio rerio; RNA Seq,GSM7812984 r1,GSM7812984,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A32_S50_L007_R1_001.fastq.gz,fastq,472785040.0,4681040.0,GSM7812984 r3,0:101,A:116503463;C:114489771;G:108989839;T:132797698;N:4269,101,,,,116503463,114489771,108989839,132797698,4269,SRX21920655,SRS19005174,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94373,,0.07285,,0.69467,,0.47913,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28151,SRR26209685,SRX21920655,SRS19005174,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 2,GSM7812984,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812984,GSM7812984: unDMSO t1 K1 2; Danio rerio; RNA Seq,GSM7812984 r1,GSM7812984,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A32_S71_L007_R1_001.fastq.gz,fastq,561056414.0,5555014.0,GSM7812984 r4,0:101,A:138240115;C:136040813;G:129592563;T:157172399;N:10524,101,,,,138240115,136040813,129592563,157172399,10524,SRX21920655,SRS19005174,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94357,,0.07109,,0.69544,,0.47169,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28152,SRR26209686,SRX21920655,SRS19005174,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 2,GSM7812984,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 2,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812984,GSM7812984: unDMSO t1 K1 2; Danio rerio; RNA Seq,GSM7812984 r1,GSM7812984,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A32_S71_L008_R1_001.fastq.gz,fastq,563109744.0,5575344.0,GSM7812984 r5,0:101,A:138778764;C:136444809;G:129964766;T:157904708;N:16697,101,,,,138778764,136444809,129964766,157904708,16697,SRX21920655,SRS19005174,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94358,,0.07253,,0.69345,,0.47443,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28153,SRR26209687,SRX21920654,SRS19005173,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 1,GSM7812983,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812983,GSM7812983: unDMSO t1 K1 1; Danio rerio; RNA Seq,GSM7812983 r1,GSM7812983,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A31_S49_L005_R1_001.fastq.gz,fastq,576725857.0,5710157.0,GSM7812983 r1,0:101,A:142557020;C:140092748;G:133378036;T:160693137;N:4916,101,,,,142557020,140092748,133378036,160693137,4916,SRX21920654,SRS19005173,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94239,,0.07259,,0.69398,,0.47675,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28154,SRR26209688,SRX21920654,SRS19005173,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 1,GSM7812983,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812983,GSM7812983: unDMSO t1 K1 1; Danio rerio; RNA Seq,GSM7812983 r1,GSM7812983,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A31_S49_L006_R1_001.fastq.gz,fastq,569166310.0,5635310.0,GSM7812983 r2,0:101,A:140702145;C:138232540;G:131558917;T:158663414;N:9294,101,,,,140702145,138232540,131558917,158663414,9294,SRX21920654,SRS19005173,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94221,,0.07252,,0.69524,,0.4833,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28155,SRR26209689,SRX21920654,SRS19005173,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 1,GSM7812983,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812983,GSM7812983: unDMSO t1 K1 1; Danio rerio; RNA Seq,GSM7812983 r1,GSM7812983,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A31_S49_L007_R1_001.fastq.gz,fastq,505699425.0,5006925.0,GSM7812983 r3,0:101,A:124940125;C:122891114;G:116919354;T:140944307;N:4525,101,,,,124940125,122891114,116919354,140944307,4525,SRX21920654,SRS19005173,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.944,,0.07242,,0.69473,,0.48289,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures
28156,SRR26209690,SRX21920654,SRS19005173,SRP463749,PRJNA1022096,Zebrafish reveal new roles for Fam83f in hatching and DNA damage mediated autophagic responses,GSE244291,Transcriptome Analysis,The FAM83 Family with sequence similarity 83 family is highly conserved in vertebrates  yet little is known of the functions of these proteins beyond a correlation with oncogenesis. Of the family  FAM83F is of particular interest because it is the only membrane targeted FAM83 protein. FAM83F has been shown to activate the canonical Wnt signalling pathway and bind to and stabilize p53 when overexpressed  two pathways often dysregulated in disease.  Insights into gene function can often be gained by studying the roles they play during development  and here we report the generation of fam83f knock out fam83f /  zebrafish  which we have used to elucidate the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the proteolytic enzyme containing hatch gland of developing zebrafish embryos  and that fam83f /  embryos hatch earlier than WT counterparts  despite developing at a comparable temporal rate. We demonstrate that fam83f /  embryos are more sensitive to ionizing radiation than WT embryos  a finding that contrasts with the previously reported role of FAM83F as a stabilizer of p53. Transcriptomic analysis shows that loss of fam83f causes downregulation of phosphatidylinositol 3 phosphate PI3P binding proteins and impairment of cellular degradation pathways  particularly autophagy  which is a crucial component of the DNA damage response. Finally  we show that Fam83f protein is itself targeted to the lysosome when expressed in cultured cells  and that this localization is dependent upon a C' terminal signal sequence.  The zebrafish lines we have generated here suggest for the first time that Fam83f plays an important role in autophagic/lysosomal processes  resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo. Overall design: fam83fa /  zebrafish are more sensitive to ionizing radiation IR than WT counterparts. We conducted bulk RNA seq on WT vs two different fam83fa /  K1 and K2 zebrafish lines by subjecting 24 hpf embryos from each genotype to IR then extracting total RNA at 2 and 10 hours following treament t1 and t2.  Three biological replicates of a minimum of 10 embryos per replicate were sequenced by bulk RNA seq to to identify any differences in the DNA damage response between WT and fam83fa /  mutants. Treatments: AD = Actinomycin D in DMSO vehicle IR = ionizing radiation 20 Grays gamma radiation unDMSO = untreated AD control vehicle only i.e. DMSO un = untreated IR control,,pubmed:39437839,,unDMSO t1 K1 1,GSM7812983,,source name:whole embryo|tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO|geo loc name:missing|collection date:missing,unDMSO t1 K1 1,Cutadapt  1.9.1 rsem 1.3.0 star 2.5.2a Assembly: GRCz11 assembly Supplementary files format and content: *.genes.results are rsem count files,whole embryo,Embryos were exposed to gamma IR of 20 Gy at xxx hpf,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,Zebrafish embryos post collection were maintained at 28.5C in E2 medium at a density ⋜  50 embryos,tissue:whole embryo|timepoint:t1|genotype:K1|treatment:unDMSO,GSM7812983,GSM7812983: unDMSO t1 K1 1; Danio rerio; RNA Seq,GSM7812983 r1,GSM7812983,1,Total RNA was extracted using RNeasy Mini Kit QIAGEN rRNA depletion Rio Zero Plus rRNA Depletion Kit Ilumina  then library prep using KAPA mRNA HyperPrep Kit for Ilumina Platforms,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP463749,,loader:fastq load.py,JON686A31_S27_L007_R1_001.fastq.gz,fastq,598624374.0,5926974.0,GSM7812983 r4,0:101,A:147863525;C:145723385;G:138724140;T:166302217;N:11107,101,,,,147863525,145723385,138724140,166302217,11107,SRX21920654,SRS19005173,SRA1722794,"Devenport, Molecular Biology, Princeton University","Devenport, Molecular Biology, Princeton University",1,0.94426,,0.07312,,0.69479,,0.48056,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,rrna_depletion,unknown,bulk,bulk,bulk,,United States,2023-09-28,Undetermined,Embryo,Whole Organism,All anatomical structures