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 5026,ERR1706529,ERX1776763,ERS1227000,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 C9,SAMEA4055890,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000051|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055890|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:24:00Z|INSDC status:public|Submitter Id:d7b0b9c0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7b0b9c0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#23,17117131,Illumina sequencing of library 17117131 constructed from sample accession ERS1227000 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence ACTGAT.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#23.cram,cram,743861850.0,4959079.0,SC RUN 20417 1#23,0:75 1:75,A:188550455;C:183533703;G:184197810;T:187506925;N:72957,75,75,,,188550455,183533703,184197810,187506925,72957,ERX1776763,ERS1227000,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96302,0.96531,0.19802,0.19492,0.73298,0.73312,0.57888,0.58087,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5027,ERR1706528,ERX1776762,ERS1226979,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 C5,SAMEA4055869,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000050|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055869|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:23:35Z|INSDC status:public|Submitter Id:d7a93fb0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7a93fb0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#22,17117119,Illumina sequencing of library 17117119 constructed from sample accession ERS1226979 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GAGTGG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#22.cram,cram,1375781700.0,9171878.0,SC RUN 20417 1#22,0:75 1:75,A:332899223;C:355056529;G:356526015;T:331162157;N:137776,75,75,,,332899223,355056529,356526015,331162157,137776,ERX1776762,ERS1226979,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96835,0.97046,0.22984,0.22663,0.753,0.75491,0.45383,0.51122,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5028,ERR1706527,ERX1776761,ERS1226967,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 C4,SAMEA4055857,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000049|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055857|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:23:22Z|INSDC status:public|Submitter Id:d7a1c5a0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7a1c5a0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#21,17117107,Illumina sequencing of library 17117107 constructed from sample accession ERS1226967 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence CGTACG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#21.cram,cram,1984245000.0,13228300.0,SC RUN 20417 1#21,0:75 1:75,A:468564557;C:523118317;G:524896332;T:467470998;N:194796,75,75,,,468564557,523118317,524896332,467470998,194796,ERX1776761,ERS1226967,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.97058,0.97302,0.22668,0.22361,0.76122,0.76319,0.6447,0.64941,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5029,ERR1706526,ERX1776760,ERS1226955,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 C1,SAMEA4055845,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000048|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055845|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:23:08Z|INSDC status:public|Submitter Id:d799d660 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d799d660 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#20,17117095,Illumina sequencing of library 17117095 constructed from sample accession ERS1226955 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GTTTCG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#20.cram,cram,1202223750.0,8014825.0,SC RUN 20417 1#20,0:75 1:75,A:288569721;C:312339594;G:312724314;T:288471735;N:118386,75,75,,,288569721,312339594,312724314,288471735,118386,ERX1776760,ERS1226955,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.9701,0.97208,0.23493,0.2308,0.75856,0.75893,0.51831,0.52827,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5030,ERR1706525,ERX1776759,ERS1226947,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B11,SAMEA4055837,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000047|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055837|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:23:00Z|INSDC status:public|Submitter Id:d7920e30 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7920e30 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#19,17117083,Illumina sequencing of library 17117083 constructed from sample accession ERS1226947 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GTGGCC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#19.cram,cram,1177752450.0,7851683.0,SC RUN 20417 1#19,0:75 1:75,A:311936666;C:276917229;G:278564226;T:310217446;N:116883,75,75,,,311936666,276917229,278564226,310217446,116883,ERX1776759,ERS1226947,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95807,0.96101,0.1671,0.16592,0.72389,0.72521,0.53692,0.53083,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5031,ERR1706524,ERX1776758,ERS1226939,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B10,SAMEA4055829,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000046|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055829|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:22:51Z|INSDC status:public|Submitter Id:d788e670 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d788e670 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#18,17117071,Illumina sequencing of library 17117071 constructed from sample accession ERS1226939 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GTGAAA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#18.cram,cram,1306412850.0,8709419.0,SC RUN 20417 1#18,0:75 1:75,A:348858023;C:304800763;G:306149463;T:346474389;N:130212,75,75,,,348858023,304800763,306149463,346474389,130212,ERX1776758,ERS1226939,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95746,0.9601,0.16534,0.1628,0.72297,0.72247,0.52807,0.52513,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5032,ERR1706523,ERX1776757,ERS1226931,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B9,SAMEA4055821,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000045|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055821|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:22:43Z|INSDC status:public|Submitter Id:d780d020 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d780d020 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#17,17117059,Illumina sequencing of library 17117059 constructed from sample accession ERS1226931 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GTCCGC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#17.cram,cram,943334550.0,6288897.0,SC RUN 20417 1#17,0:75 1:75,A:250576062;C:221289820;G:222449063;T:248930088;N:89517,75,75,,,250576062,221289820,222449063,248930088,89517,ERX1776757,ERS1226931,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95898,0.9615,0.16669,0.16433,0.72636,0.7262,0.53064,0.52544,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5033,ERR1706522,ERX1776756,ERS1226910,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B6,SAMEA4055800,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000044|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055800|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:22:20Z|INSDC status:public|Submitter Id:d778e0e0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d778e0e0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#16,17117142,Illumina sequencing of library 17117142 constructed from sample accession ERS1226910 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence CCGTCC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#16.cram,cram,1397786700.0,9318578.0,SC RUN 20417 1#16,0:75 1:75,A:370541493;C:328697895;G:330649047;T:367761875;N:136390,75,75,,,370541493,328697895,330649047,367761875,136390,ERX1776756,ERS1226910,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95779,0.96097,0.15701,0.15529,0.72462,0.72425,0.53437,0.53651,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5034,ERR1706521,ERX1776755,ERS1226903,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B4,SAMEA4055793,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000043|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055793|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:22:11Z|INSDC status:public|Submitter Id:d7713fc0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7713fc0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#15,17117130,Illumina sequencing of library 17117130 constructed from sample accession ERS1226903 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence ATGTCA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#15.cram,cram,2090642550.0,13937617.0,SC RUN 20417 1#15,0:75 1:75,A:573574606;C:473115476;G:474137164;T:569607223;N:208081,75,75,,,573574606,473115476,474137164,569607223,208081,ERX1776755,ERS1226903,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95314,0.95602,0.13917,0.13779,0.71605,0.7161,0.5009,0.49877,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5035,ERR1706520,ERX1776754,ERS1226896,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B3,SAMEA4055786,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000042|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055786|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:22:03Z|INSDC status:public|Submitter Id:d7697790 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7697790 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#14,17117118,Illumina sequencing of library 17117118 constructed from sample accession ERS1226896 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence AGTTCC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#14.cram,cram,1829182200.0,12194548.0,SC RUN 20417 1#14,0:75 1:75,A:501496169;C:412501773;G:416194477;T:498811651;N:178130,75,75,,,501496169,412501773,416194477,498811651,178130,ERX1776754,ERS1226896,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95118,0.95498,0.14818,0.14602,0.71431,0.71478,0.49278,0.4993,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5036,ERR1706519,ERX1776753,ERS1226884,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B2,SAMEA4055774,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000041|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055774|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:21:52Z|INSDC status:public|Submitter Id:d761fd80 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d761fd80 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#13,17117106,Illumina sequencing of library 17117106 constructed from sample accession ERS1226884 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence AGTCAA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#13.cram,cram,969474450.0,6463163.0,SC RUN 20417 1#13,0:75 1:75,A:262909712;C:222419636;G:222543360;T:261505885;N:95857,75,75,,,262909712,222419636,222543360,261505885,95857,ERX1776753,ERS1226884,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95449,0.95866,0.15114,0.14937,0.71437,0.71465,0.51271,0.50858,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5037,ERR1706518,ERX1776752,ERS1226870,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 B1,SAMEA4055760,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000040|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055760|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:21:36Z|INSDC status:public|Submitter Id:d75a3550 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish actin:prl3.1 embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d75a3550 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#12,17117094,Illumina sequencing of library 17117094 constructed from sample accession ERS1226870 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence CTTGTA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#12.cram,cram,1454862600.0,9699084.0,SC RUN 20417 1#12,0:75 1:75,A:398496982;C:328471618;G:330736633;T:397014742;N:142625,75,75,,,398496982,328471618,330736633,397014742,142625,ERX1776752,ERS1226870,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95557,0.95961,0.13233,0.1312,0.71441,0.71488,0.49023,0.48445,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5038,ERR1706517,ERX1776751,ERS1226860,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A12,SAMEA4055750,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000039|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055750|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:21:28Z|INSDC status:public|Submitter Id:d7529430 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7529430 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#11,17117082,Illumina sequencing of library 17117082 constructed from sample accession ERS1226860 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GGCTAC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#11.cram,cram,1746393900.0,11642626.0,SC RUN 20417 1#11,0:75 1:75,A:431979433;C:441330253;G:441743262;T:431172392;N:168560,75,75,,,431979433,441330253,441743262,431172392,168560,ERX1776751,ERS1226860,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96506,0.9668,0.22176,0.21891,0.74491,0.74621,0.60192,0.59716,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5039,ERR1706516,ERX1776750,ERS1226849,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A11,SAMEA4055739,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000038|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055739|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:21:18Z|INSDC status:public|Submitter Id:d74a56d0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d74a56d0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#10,17117070,Illumina sequencing of library 17117070 constructed from sample accession ERS1226849 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence TAGCTT.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#10.cram,cram,1489471050.0,9929807.0,SC RUN 20417 1#10,0:75 1:75,A:370582953;C:374410965;G:374249460;T:370079632;N:148040,75,75,,,370582953,374410965,374249460,370079632,148040,ERX1776750,ERS1226849,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96529,0.96757,0.21607,0.21231,0.74272,0.7432,0.59703,0.6019,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5040,ERR1706515,ERX1776749,ERS1226835,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A9,SAMEA4055725,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000037|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055725|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:21:06Z|INSDC status:public|Submitter Id:d7428ea0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7428ea0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#9,17117058,Illumina sequencing of library 17117058 constructed from sample accession ERS1226835 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GATCAG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#9.cram,cram,812740200.0,5418268.0,SC RUN 20417 1#9,0:75 1:75,A:212802181;C:193901585;G:194345487;T:211612018;N:78929,75,75,,,212802181,193901585,194345487,211612018,78929,ERX1776749,ERS1226835,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95783,0.95996,0.17262,0.1685,0.72413,0.72448,0.53944,0.53954,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5041,ERR1706514,ERX1776748,ERS1226824,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A8,SAMEA4055714,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000036|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055714|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:58Z|INSDC status:public|Submitter Id:d73ac670 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d73ac670 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#8,17117141,Illumina sequencing of library 17117141 constructed from sample accession ERS1226824 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence ACTTGA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#8.cram,cram,1782401400.0,11882676.0,SC RUN 20417 1#8,0:75 1:75,A:458909593;C:432851102;G:434061292;T:456406952;N:172461,75,75,,,458909593,432851102,434061292,456406952,172461,ERX1776748,ERS1226824,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96096,0.96367,0.18551,0.18292,0.73413,0.73373,0.56474,0.56438,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5042,ERR1706513,ERX1776747,ERS1226813,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A7,SAMEA4055703,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000035|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055703|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:49Z|INSDC status:public|Submitter Id:d7332550 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7332550 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#7,17117129,Illumina sequencing of library 17117129 constructed from sample accession ERS1226813 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence CAGATC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#7.cram,cram,396397350.0,2642649.0,SC RUN 20417 1#7,0:75 1:75,A:103468605;C:95292253;G:94558692;T:103038245;N:39555,75,75,,,103468605,95292253,94558692,103038245,39555,ERX1776747,ERS1226813,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96013,0.96276,0.17171,0.16938,0.72941,0.73046,0.54526,0.54527,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 5043,ERR1706512,ERX1776746,ERS1226790,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A6,SAMEA4055680,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000034|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055680|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:34Z|INSDC status:public|Submitter Id:d72b5d20 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d72b5d20 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#6,17117117,Illumina sequencing of library 17117117 constructed from sample accession ERS1226790 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence GCCAAT.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#6.cram,cram,652493400.0,4349956.0,SC RUN 20417 1#6,,,,,,,,,,,,ERX1776746,ERS1226790,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.94432,0.94163,0.16831,0.16496,0.72912,0.72766,0.50382,0.5065,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5044,ERR1706511,ERX1776745,ERS1226779,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A5,SAMEA4055669,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000033|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055669|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:26Z|INSDC status:public|Submitter Id:d7219920 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7219920 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#5,17117105,Illumina sequencing of library 17117105 constructed from sample accession ERS1226779 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence ACAGTG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#5.cram,cram,825205650.0,5501371.0,SC RUN 20417 1#5,0:75 1:75,A:224928393;C:188238841;G:188933086;T:223024865;N:80465,75,75,,,224928393,188238841,188933086,223024865,80465,ERX1776745,ERS1226779,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95709,0.96033,0.14048,0.13863,0.71887,0.71987,0.49099,0.49207,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5045,ERR1706510,ERX1776744,ERS1226767,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A4,SAMEA4055657,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000032|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055657|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:16Z|INSDC status:public|Submitter Id:d71934b0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d71934b0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#4,17117093,Illumina sequencing of library 17117093 constructed from sample accession ERS1226767 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence TGACCA.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#4.cram,cram,767189250.0,5114595.0,SC RUN 20417 1#4,0:75 1:75,A:210673766;C:173125942;G:174062852;T:209253897;N:72793,75,75,,,210673766,173125942,174062852,209253897,72793,ERX1776744,ERS1226767,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95581,0.95773,0.11947,0.11773,0.72103,0.72178,0.4674,0.46869,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5046,ERR1706509,ERX1776743,ERS1226757,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A3,SAMEA4055647,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000031|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055647|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:20:08Z|INSDC status:public|Submitter Id:d7092f20 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d7092f20 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#3,17117081,Illumina sequencing of library 17117081 constructed from sample accession ERS1226757 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence TTAGGC.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#3.cram,cram,776002350.0,5173349.0,SC RUN 20417 1#3,0:75 1:75,A:207553748;C:180473885;G:180618978;T:207278008;N:77731,75,75,,,207553748,180473885,180618978,207278008,77731,ERX1776743,ERS1226757,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.96457,0.96611,0.23757,0.23414,0.74507,0.74497,0.55158,0.55086,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5047,ERR1706508,ERX1776742,ERS1226745,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A2,SAMEA4055635,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000030|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055635|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:19:59Z|INSDC status:public|Submitter Id:d6f77be0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d6f77be0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#2,17117069,Illumina sequencing of library 17117069 constructed from sample accession ERS1226745 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence CGATGT.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#2.cram,cram,581391900.0,3875946.0,SC RUN 20417 1#2,0:75 1:75,A:156042125;C:134966129;G:135462829;T:154862718;N:58099,75,75,,,156042125,134966129,135462829,154862718,58099,ERX1776742,ERS1226745,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.9582,0.96234,0.1297,0.12747,0.72863,0.72949,0.48838,0.48132,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 5048,ERR1706507,ERX1776741,ERS1226722,ERP016002,PRJEB14364,Transcriptome profiling of zebrafish actin prl3 1 transgenic embryos,Transcriptome_profiling_of_zebrafish_actin_prl3_1_transgenic_embryos-sc-4266,Transcriptome Analysis,RNA Seq data was generated from RNA of zebrafish actin:prl3.1 transgenic embryos for transcriptional profiling.,ArrayExpress:E ERAD 507,,,zmp ph260 A1,SAMEA4055612,Wellcome Sanger Institute,ArrayExpress DevelopmentalStage:Segmentation:26+somites Pharyngula:Prim 5 ZFS:0000028 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA first public:2016 10 24|ENA last update:2016 06 28|External Id:SAMEA4055612|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2016 10 24T16:33:40Z|INSDC last update:2016 06 28T15:19:38Z|INSDC status:public|Submitter Id:d6e3f3e0 2e55 11e6 a63e 3c4a9275d6c8|common name:zebrafish|sample description:RNA from a single zebrafish non transgenic actin:prl3.1 sibling embryo plus ERCC spike mix 2 Ambion. The sample has been DNAse treated.|sample name:d6e3f3e0 2e55 11e6 a63e 3c4a9275d6c8|strain:mixed,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 20417 1#1,17117057,Illumina sequencing of library 17117057 constructed from sample accession ERS1226722 for study accession ERP016002. This is part of an Illumina multiplexed sequencing run 20417 1. This submission includes reads tagged with the sequence ATCACG.,RNA seq dUTP eukaryotic,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP016002,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2016 10 24|ENA LAST UPDATE:2018 11 16,20417_1#1.cram,cram,1233271650.0,8221811.0,SC RUN 20417 1#1,0:75 1:75,A:334336992;C:282740616;G:283953463;T:332113804;N:126775,75,75,,,334336992,282740616,283953463,332113804,126775,ERX1776741,ERS1226722,ERA740537,European Nucleotide Archive,Wellcome Sanger Institute,2,0.95579,0.95701,0.15941,0.15689,0.71758,0.71709,0.4967,0.5163,75,75,B,B,biological fallback assumption,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2016-06-28,Multi-stage,Embryo,Whole Organism,All anatomical structures 9892,ERR4172795,ERX4136409,ERS4580819,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sibling 3,SAMEA6853229,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853229|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sibling 3|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|sample name:E MTAB 9113:sibling 3|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sibling 3 p,sibling 3 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-sibs-3_S6_R1.fastq.gz UCLGNS1141-gfp-sibs-3_S6_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp sibs 3 S6 R,0:81 1:81,A:1144578958;C:1113109875;G:1138809559;T:1117038434;N:213932,81,81,,,1144578958,1113109875,1138809559,1117038434,213932,ERX4136409,ERS4580819,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.96684,0.96492,0.03244,0.0317,0.71236,0.71514,0.45871,0.46189,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 9893,ERR4172794,ERX4136408,ERS4580818,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sibling 2,SAMEA6853228,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853228|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sibling 2|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|sample name:E MTAB 9113:sibling 2|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sibling 2 p,sibling 2 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-sibs-2_S4_R1.fastq.gz UCLGNS1141-gfp-sibs-2_S4_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp sibs 2 S4 R,0:81 1:81,A:1056280368;C:1036355469;G:1045429486;T:1035773622;N:189389,81,81,,,1056280368,1036355469,1045429486,1035773622,189389,ERX4136408,ERS4580818,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.95511,0.95696,0.02941,0.02903,0.71492,0.71628,0.45765,0.46537,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 9894,ERR4172793,ERX4136407,ERS4580817,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sibling 1,SAMEA6853227,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853227|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sibling 1|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|sample name:E MTAB 9113:sibling 1|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sibling 1 p,sibling 1 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-sibs-1_S2_R1.fastq.gz UCLGNS1141-gfp-sibs-1_S2_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp sibs 1 S2 R,0:81 1:81,A:1121176859;C:1093927654;G:1108608188;T:1101082277;N:209264,81,81,,,1121176859,1093927654,1108608188,1101082277,209264,ERX4136407,ERS4580817,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.95795,0.96004,0.02885,0.02854,0.71648,0.71756,0.44348,0.43956,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 9895,ERR4172792,ERX4136406,ERS4580816,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sfpq 3,SAMEA6853226,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853226|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 3|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 3|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sfpq 3 p,sfpq 3 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:sfpq / ,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-neg_3_S5_R1.fastq.gz UCLGNS1141-gfp-neg_3_S5_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp neg 3 S5 R,0:81 1:81,A:845849249;C:772302798;G:911660213;T:787409272;N:154042,81,81,,,845849249,772302798,911660213,787409272,154042,ERX4136406,ERS4580816,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.96298,0.95486,0.03415,0.03476,0.7167,0.73503,0.4665,0.45947,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 9896,ERR4172791,ERX4136405,ERS4580815,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sfpq 2,SAMEA6853225,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853225|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 2|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 2|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sfpq 2 p,sfpq 2 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:sfpq / ,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-neg-2_S3_R1.fastq.gz UCLGNS1141-gfp-neg-2_S3_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp neg 2 S3 R,0:81 1:81,A:961156886;C:917011292;G:942713581;T:936109877;N:157724,81,81,,,961156886,917011292,942713581,936109877,157724,ERX4136405,ERS4580815,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.96276,0.96147,0.03436,0.03382,0.71892,0.72021,0.4581,0.46402,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 9897,ERR4172790,ERX4136404,ERS4580814,ERP121885,PRJEB38455,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E-MTAB-9113,Transcriptome Analysis,RNA was extracted from whole zebrafish embryos at 24 hpf to examine changes in gene expression and splicing in sfpq null mutants,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 05 22,,Protocols: Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,sfpq 1,SAMEA6853224,Centre for Developmental Neurobiology King's College London,ENA FIRST PUBLIC:2020 12 01T04:08:08Z|ENA LAST UPDATE:2020 05 22T17:13:15Z|External Id:SAMEA6853224|INSDC center name:Centre for Developmental Neurobiology King's College London|INSDC first public:2020 12 01T04:08:08Z|INSDC last update:2020 05 22T17:13:15Z|INSDC status:public|Submitter Id:E MTAB 9113:sfpq 1|age:24|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:sfpq / |sample name:E MTAB 9113:sfpq 1|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,E MTAB 9113:sfpq 1 p,sfpq 1 p,RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,Embryos were collected from in crosses of sfpq+/ adult zebrafish RNA was extracted using the RNEasy Mini Kit Qiagen Library constructed with ribodepletion,Experimental Factor: genotype:sfpq / ,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,ERP121885,Illumina HiSeq 2500 paired end sequencing; RNA seq of sfpq / zebrafish embryos and siblings at 24 hpf,ENA FIRST PUBLIC:2021 01 27|ENA LAST UPDATE:2020 06 19,UCLGNS1141-gfp-neg-1_S1_R1.fastq.gz UCLGNS1141-gfp-neg-1_S1_R2.fastq.gz,fastq fastq,,,E MTAB 9113:UCLGNS1141 gfp neg 1 S1 R,0:81 1:81,A:1046317524;C:1014946275;G:1036910299;T:1023619857;N:185237,81,81,,,1046317524,1014946275,1036910299,1023619857,185237,ERX4136404,ERS4580814,ERA2625401,Centre for Developmental Neurobiology King,Centre for Developmental Neurobiology King,2,0.96015,0.96173,0.03114,0.03098,0.7175,0.71865,0.46666,0.46399,81,81,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,United Kingdom,2020-05-22,Multi-stage,Multi-stage,Embryo Imprecise,All anatomical structures 11130,ERR9979395,ERX9520370,ERS12499848,ERP139765,PRJEB54901,Single cell RNA sequencing of germ free zebrafish embryos,E-MTAB-11984,Transcriptome Analysis,The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,,Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Sample 2 WT GO+BA,SAMEA110401656,"Institute of Environmental Medicine, Karolinska Institutet",ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,E MTAB 11984:Sample 2 WT GO+BA p,Sample 2 WT GO+BA p,Single cell RNA sequencing of germ free zebrafish embryos,The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM,AMPLICON,TRANSCRIPTOMIC SINGLE CELL,size fractionation,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP139765,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,P22202_7002_S2_L002_R1_001.fastq.gz P22202_7002_S2_L002_R2_001.fastq.gz,fastq fastq,18144406998.0,153766161.0,E MTAB 11984:P22202 7002 S2 L002,0:28 1:90,A:5277424121;C:3880928155;G:4118706097;T:4866649878;N:698747,28,90,,,5277424121,3880928155,4118706097,4866649878,698747,ERX9520370,ERS12499848,ERA16483151,"Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive","Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive",2,0.0109,0.90145,0.0053,0.21373,0.98746,0.79423,0.41198,0.54447,28,90,T,B,sc-like readlen,illumina,novaseq_era,full_length,size_fractionation,trueseq,sc,single_cell_droplet,10x,,Sweden,2022-07-22,Multi-stage,Embryo,Whole Organism,All anatomical structures 11131,ERR9979394,ERX9520370,ERS12499848,ERP139765,PRJEB54901,Single cell RNA sequencing of germ free zebrafish embryos,E-MTAB-11984,Transcriptome Analysis,The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,,Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Sample 2 WT GO+BA,SAMEA110401656,"Institute of Environmental Medicine, Karolinska Institutet",ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401656|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 2 WT GO+BA|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 2 WT GO+BA|sex:not available|stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM|strain:AB,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,E MTAB 11984:Sample 2 WT GO+BA p,Sample 2 WT GO+BA p,Single cell RNA sequencing of germ free zebrafish embryos,The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Experimental Factor: stimulus:graphene oxide 30 ug/mL and butyrate 2.5 mM,AMPLICON,TRANSCRIPTOMIC SINGLE CELL,size fractionation,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP139765,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,P22202_7002_S2_L001_R1_001.fastq.gz P22202_7002_S2_L001_R2_001.fastq.gz,fastq fastq,18458951574.0,156431793.0,E MTAB 11984:P22202 7002 S2 L001,0:28 1:90,A:5372920926;C:3946687814;G:4185647697;T:4953205454;N:489683,28,90,,,5372920926,3946687814,4185647697,4953205454,489683,ERX9520370,ERS12499848,ERA16483151,"Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive","Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive",2,0.01037,0.90168,0.0048,0.21246,0.98752,0.79297,0.42601,0.54996,28,90,T,B,sc-like readlen,illumina,novaseq_era,full_length,size_fractionation,trueseq,sc,single_cell_droplet,10x,,Sweden,2022-07-22,Multi-stage,Embryo,Whole Organism,All anatomical structures 11132,ERR9979392,ERX9520369,ERS12499847,ERP139765,PRJEB54901,Single cell RNA sequencing of germ free zebrafish embryos,E-MTAB-11984,Transcriptome Analysis,The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,,Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Sample 1 WT control,SAMEA110401655,"Institute of Environmental Medicine, Karolinska Institutet",ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,E MTAB 11984:Sample 1 WT control p,Sample 1 WT control p,Single cell RNA sequencing of germ free zebrafish embryos,The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Experimental Factor: stimulus:n1,AMPLICON,TRANSCRIPTOMIC SINGLE CELL,size fractionation,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP139765,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,P22202_7001_S1_L001_R1_001.fastq.gz P22202_7001_S1_L001_R2_001.fastq.gz,fastq fastq,19396994226.0,164381307.0,E MTAB 11984:P22202 7001 S1 L001,0:28 1:90,A:5665199484;C:4091196594;G:4415211076;T:5224866754;N:520318,28,90,,,5665199484,4091196594,4415211076,5224866754,520318,ERX9520369,ERS12499847,ERA16483151,"Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive","Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive",2,0.01093,0.89238,0.00557,0.232,0.98764,0.79444,0.40806,0.54781,28,90,T,B,sc-like readlen,illumina,novaseq_era,full_length,size_fractionation,trueseq,sc,single_cell_droplet,10x,,Sweden,2022-07-22,Multi-stage,Embryo,Whole Organism,All anatomical structures 11133,ERR9979393,ERX9520369,ERS12499847,ERP139765,PRJEB54901,Single cell RNA sequencing of germ free zebrafish embryos,E-MTAB-11984,Transcriptome Analysis,The present study was conducted in the frame of the EU funded Graphene Flagship project. We previously evaluated the impact of graphene oxide GO on the gut microbiome in adult zebrafish by performing 16S rRNA gene sequencing in wild type versus AhR deficient zebrafish. Here we performed single cell RNA sequencing 10x Genomics on whole dissociated germ free GF zebrafish embryos exposed at 5 dpf to GO plus the microbial metabolite butyrate to gain insight into the impact on specific cell populations in GF zebrafish.,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,,Protocols: The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Sample 1 WT control,SAMEA110401655,"Institute of Environmental Medicine, Karolinska Institutet",ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22|External Id:SAMEA110401655|INSDC center alias:Institute of Environmental Medicine Karolinska Institutet|INSDC center name:Institute of Environmental Medicine Karolinska Institutet|INSDC first public:2022 07 22T16:26:50Z|INSDC last update:2022 07 22T16:26:50Z|INSDC status:public|Submitter Id:E MTAB 11984:Sample 1 WT control|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:embryo|genotype:wild type genotype|individual:pool of 80 individuals|isolate:not applicable|organism part:whole organism|sample name:E MTAB 11984:Sample 1 WT control|sex:not available|stimulus:n1|strain:AB,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,E MTAB 11984:Sample 1 WT control p,Sample 1 WT control p,Single cell RNA sequencing of germ free zebrafish embryos,The generation of germ free zebrafish wild type AB followed previously established protocols [1]. In brief 2 hpf embryos were transferred to petri dishes with sterile E3 medium supplemented with ampicillin 100 μg/mL kanamycin 5 μg/mL and amphotericin B 250 ng/mL and incubated at 28°C. At 50% epiboly up to shield stage 6 hpf the embryos were surface disinfected with 0.1% of polyvinylpyrroidone iodine PVP I for exactly 2 min followed by 0.003% sterile bleach immersion for 18 min. The embryos were then rinsed with sterile E3 medium transferred to flasks and incubated at 28°C. The viability was monitored and sterile medium was refreshed daily. At day 4 the hatched embryos were used for the sterility validation. A day 5 germ free zebrafish were exposed to the combination of GO 30 µg/mL and butyrate 2.5 mM for 24 h. GO and BA were pre incubated for 1 h prior to the exposure. Twenty larvae were used as one replicate four replicates i.e. eighty larvae in total were used for each condition. post the exposure zebrafish larvae were dissociated for single cell suspension following the published protocol [2]. Briefly zebrafish larvae were euthanized with 0.01% of tricaine for 5 min collected in 1.5 mL tube and washed with PBS for three times. The dissociation was initiated by adding 500 μL of pre warmed enzyme mix containing 460 μL of 0.25% trypsin EDTA Gibco and 40 μL of collagenase Sigma Aldrich at the concentration of 100 mg/mL followed by mechanical dissociation using P1000 and then P200 pipette tips in heat block at 30 °C until the tissue was no longer visible about 10 min. The dissociation was then stopped by adding 800 μL DMEM Corning Cat# 10 013 CV with 10% FBS Fisher Scientific. The cell pellets were collected by centrifuging at 1000 rpm for 5 min at RT followed by washing with PBS. The cells were next resuspended in 0.5 mL DMEM with 10% FBS. Four replicates from each condition were pooled together at this step and filtered through 40 μm nylon mesh with an additional washing using DMEM with 10% FBS. The cell suspension was then stained with a fluorescent DNA dye DRAQ7 to exclude non viable cells Invitrogen Cat# D15106 at the dose of 3 μM for 10 min at room temperature before the fluorescence activated cell sorting BD FACSAria III BD Biosciences NJ USA. The sorting was performed using a 100 microns nozzle with the flow rate of 2 and temperature control at 4 °C and gated based on forward scatter and DRAQ7. The DRAQ7 negative cells of each sample were FACS sorted into the tubes containing DMEM with 10% FBS and placed on ice immediately post the sorting. The cells were then proceeded with the single cell RNA sequencing analysis. 1. Pham LN Kanther M Semova I & Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3 1862 1875 2008. 2. Bresciani E Broadbridge E Liu PP An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae MethodsX. 5 1287 1290 2018. Single cell RNA sequencing scRNA seq was performed by Eukaryotic Single Cell Genomics Facility at SciLifeLab Stockholm. The single cells collected from FACS sorting were loaded on a 10x GemCode Single Cell Instrument 10x Genomics Pleasanton USA to generate single cell gel beads in emulsion GEMs using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics following the manufacture's protocol. Briefly GEMs were generated by combining barcoded Single Cell three prime v3.1 Gel Beads a Master Mix containing cells and Partitioning Oil onto Chromium Next GEM Chip G. Following GEMs generation the Gel Bead was dissolved primers containing an Illumina TruSeq Read 1read 1 sequencing primer 16 nt 10x Barcode 12 nt unique molecular identifier UMI and 30 nt polydT sequence were released and the co partitioned cells were lysed. The nucleic acid library construction was performed using the Chromium Next GEM Single Cell three prime GEM Library & Gel Bead Kit v3.1 10x Genomics 16 rxns PN 1000268 following the manufacture's protocol. post GEMs generation primers were mixed with the cell lysate and a Master Mix containing reverse transcription RT reagents to produce barcoded full length cDNA from polyadenylated mRNA. Silane magnetic beads were used to purify the first strand cDNA from the post GEM RT reaction mixture which included leftover biochemical reagents and primers. Barcoded full length cDNA was amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. P5 P7 i7 and i5 sample indexes and TruSeq Read 2 read 2 primer sequence were added via End Repair A tailing Adaptor Ligation and PCR. The final libraries containing the P5 and P7 primers used in Illumina amplification were prepared for an estimated 5000 nuclei per sample.,Experimental Factor: stimulus:n1,AMPLICON,TRANSCRIPTOMIC SINGLE CELL,size fractionation,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP139765,Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of germ free zebrafish embryos,ENA FIRST PUBLIC:2022 07 22|ENA LAST UPDATE:2022 07 22,P22202_7001_S1_L002_R1_001.fastq.gz P22202_7001_S1_L002_R2_001.fastq.gz,fastq fastq,19063958224.0,161558968.0,E MTAB 11984:P22202 7001 S1 L002,0:28 1:90,A:5563588848;C:4022930643;G:4344549562;T:5132136743;N:752428,28,90,,,5563588848,4022930643,4344549562,5132136743,752428,ERX9520369,ERS12499847,ERA16483151,"Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive","Institute of Environmental Medicine, Karolinska Institutet|European Nucleotide Archive",2,0.01118,0.89308,0.00573,0.23128,0.98737,0.79354,0.39264,0.54552,28,90,T,B,sc-like readlen,illumina,novaseq_era,full_length,size_fractionation,trueseq,sc,single_cell_droplet,10x,,Sweden,2022-07-22,Multi-stage,Embryo,Whole Organism,All anatomical structures 11238,ERR10782555,ERX10233132,ERS14439197,ERP144048,PRJEB58983,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E-MTAB-12503,Transcriptome Analysis,Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Sample 6,E MTAB 12503:Sample 6,,strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 6|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,,,,,,,,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E MTAB 12503:Sample 6 p,Sample 6 p,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,PAIRED,ILLUMINA,Illumina HiSeq 4000,,ERP144048,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,20170530.A-6_R1.fastq.gz 20170530.A-6_R2.fastq.gz,fastq fastq,14809693138.0,49038719.0,E MTAB 12503:20170530.A 6 R,0:151 1:151,A:4069749497;C:3367864217;G:3430269434;T:3928390721;N:13419269,151,151,,,4069749497,3367864217,3430269434,3928390721,13419269,ERX10233132,ERS14439197,ERA20162442,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,2,0.83945,0.69977,0.27269,0.22488,0.74523,0.77654,0.4899,0.4358,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,trueseq,sc,unknown,unknown,,Switzerland,2023-03-31,Multi-stage,Embryo,Whole Organism,All anatomical structures 11239,ERR10782554,ERX10233131,ERS14439196,ERP144048,PRJEB58983,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E-MTAB-12503,Transcriptome Analysis,Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Sample 5,E MTAB 12503:Sample 5,,strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 5|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:wild type genotype|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,,,,,,,,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E MTAB 12503:Sample 5 p,Sample 5 p,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Experimental Factor: developmental stage:bud|Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,PAIRED,ILLUMINA,Illumina HiSeq 4000,,ERP144048,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,20170530.A-5_R1.fastq.gz 20170530.A-5_R2.fastq.gz,fastq fastq,14954968728.0,49519764.0,E MTAB 12503:20170530.A 5 R,0:151 1:151,A:4063340262;C:3440933120;G:3455723079;T:3981396987;N:13575280,151,151,,,4063340262,3440933120,3455723079,3981396987,13575280,ERX10233131,ERS14439196,ERA20162442,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,2,0.85891,0.86859,0.28842,0.28866,0.7349,0.75051,0.45532,0.49145,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,trueseq,sc,unknown,unknown,,Switzerland,2023-03-31,Multi-stage,Embryo,Whole Organism,All anatomical structures 11242,ERR10782551,ERX10233128,ERS14439193,ERP144048,PRJEB58983,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E-MTAB-12503,Transcriptome Analysis,Defects in blood development are a major contributor to complex congenital anomalies. Thrombocytopenia Absent Radius TAR Syndrome is a rare congenital disease presenting with reduced blood platelets megakaryocytic thrombocytopenia and forelimb anomalies concurrent with variable heart and kidney defects caused by hypomorphic RBM8A/Y14 gene function. RBM8A encodes a component of the ubiquitous exon junction complex involved in mRNA splicing transport and nonsense mediated decay. How perturbing a general mRNA processing factor causes the distinct phenotypes of TAR Syndrome remains unknown. Here we connect zebrafish rbm8a perturbation to early hematopoiesis defects via attenuated planar cell polarity PCP signaling involved in controlling developmental cell arrangements. Combining different genetic means to reduce rbm8a function we find a significant reduction of cd41 positive thrombocytes in hypomorphic rbm8a larvae. Transcriptomics analysis documents rbm8a mutant zebrafish embryos already post gastrulation accumulate mRNAs with erroneously included introns a hallmark of defective nonsense mediated decay. Affected mRNAs include transcripts encoding components of the non canonical Wnt pathway involved in PCP signaling and rbm8a mutant embryos show hallmarks of Wnt/PCP dependent early convergent extension defects. We establish that reduced rbm8a function synergizes with perturbations in Wnt/PCP pathway genes including wnt5b wnt11f2 fzd7a and vangl2. Following axis formation rbm8a perturbation impairs the migration and architecture of the lateral plate mesoderm LPM that forms the hematopoietic cardiovascular kidney and forelimb skeleton progenitors. Subsequently rbm8a perturbation impairs expression of early hematopoietic/endothelial genes including runx1a kdrl sox7 and the megakaryocyte regulator gfi1aa. Lastly we document similar hematopoietic defects upon loss of vangl2. Together our data link reduced rbm8a function to LPM migration and hematopoietic defects via attenuated Wnt/PCP signaling. Our findings establish a developmental framework that connects the complex TAR Syndrome phenotypes to a potential LPM origin.,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,Protocols: Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Sample 7,E MTAB 12503:Sample 7,,strain:mixed AB and Tubingen|ENA FIRST PUBLIC:2023 03 31|individual:20170530.A 7|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 03 31|scientific name:Danio rerio|common name:zebrafish|organism part:embryo|age:10|developmental stage:bud|genotype:rbm8a d5/d5|ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,,,,,,,,,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,E MTAB 12503:Sample 7 p,Sample 7 p,RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,Zebrafish embryos were collected at indicated developmental stages tailbud stage and 24 hpf. Total RNA was isolated from genotype defined pooled embryos by Trizol LS extraction as per manufacturer’s guidelines Invitrogen with final precipitation using 70% Ethanol. Libraries for sequencing were generated with the TruSeq RNA Sample Preparation kit Illumina.,Experimental Factor: developmental stage:bud|Experimental Factor: genotype:rbm8a d5/d5,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,PAIRED,ILLUMINA,Illumina HiSeq 4000,,ERP144048,Illumina HiSeq 4000 paired end sequencing; RNA seq of zebrafish embryos to investigate how Rbm8a deficiency akin to TAR Syndrome causes hematopoietic defects by modulating Wnt/PCP signaling,ENA FIRST PUBLIC:2023 03 31|ENA LAST UPDATE:2023 03 31,20170530.A-7_R1.fastq.gz 20170530.A-7_R2.fastq.gz,fastq fastq,12726557236.0,42140918.0,E MTAB 12503:20170530.A 7 R,0:151 1:151,A:3510176970;C:2879679978;G:2932244172;T:3392911734;N:11544382,151,151,,,3510176970,2879679978,2932244172,3392911734,11544382,ERX10233128,ERS14439193,ERA20162442,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,2,0.88531,0.88678,0.33279,0.3329,0.73545,0.74992,0.52548,0.52864,151,151,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,trueseq,sc,unknown,unknown,,Switzerland,2023-03-31,Multi-stage,Embryo,Whole Organism,All anatomical structures 11821,ERR11834690,ERX11232843,ERS16254762,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 4,SAMEA114265804,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265804|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 4|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:9.6|sample name:E MTAB 13263:Sample 4|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 4 p,Sample 4 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2113_dre_C1_clobetasolpropionate_lib667285_10169_3_2.fastq.gz NG-32417_R2113_dre_C1_clobetasolpropionate_lib667285_10169_3_1.fastq.gz,fastq fastq,9764006696.0,32331148.0,E MTAB 13263:NG 32417 R2113 dre C1 clobetasolpropionate lib667285 10169 3 ,0:151 1:151,A:2627275848;C:2223323771;G:2323190525;T:2590103027;N:113525,151,151,,,2627275848,2223323771,2323190525,2590103027,113525,ERX11232843,ERS16254762,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95424,0.95502,0.12596,0.12401,0.65884,0.65825,0.48134,0.48327,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11822,ERR11834689,ERX11232842,ERS16254761,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 3,SAMEA114265803,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265803|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 3|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.4|sample name:E MTAB 13263:Sample 3|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 3 p,Sample 3 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2112_dre_nc_clobetasolpropionate_lib673071_10180_1_2.fastq.gz NG-32417_R2112_dre_nc_clobetasolpropionate_lib673071_10180_1_1.fastq.gz,fastq fastq,13326663550.0,44128025.0,E MTAB 13263:NG 32417 R2112 dre nc clobetasolpropionate lib673071 10180 1 ,0:151 1:151,A:3641640344;C:3020489123;G:3072643196;T:3591659412;N:231475,151,151,,,3641640344,3020489123,3072643196,3591659412,231475,ERX11232842,ERS16254761,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95271,0.95293,0.11945,0.1151,0.65819,0.6579,0.47257,0.47803,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11823,ERR11834684,ERX11232837,ERS16254756,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 1,SAMEA114265798,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265798|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 1|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.3|sample name:E MTAB 13263:Sample 1|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 1 p,Sample 1 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2110_dre_nc_clobetasolpropionate_lib667282_10169_3_1.fastq.gz NG-32417_R2110_dre_nc_clobetasolpropionate_lib667282_10169_3_2.fastq.gz,fastq fastq,10152616370.0,33617935.0,E MTAB 13263:NG 32417 R2110 dre nc clobetasolpropionate lib667282 10169 3 ,0:151 1:151,A:2743478864;C:2319134955;G:2378048842;T:2711839167;N:114542,151,151,,,2743478864,2319134955,2378048842,2711839167,114542,ERX11232837,ERS16254756,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95273,0.95457,0.13567,0.13174,0.65896,0.65841,0.47947,0.47815,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11824,ERR11834691,ERX11232844,ERS16254763,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 5,SAMEA114265805,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265805|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 5|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:9.2|sample name:E MTAB 13263:Sample 5|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 5 p,Sample 5 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2114_dre_C1_clobetasolpropionate_lib667286_10169_3_1.fastq.gz NG-32417_R2114_dre_C1_clobetasolpropionate_lib667286_10169_3_2.fastq.gz,fastq fastq,11690304334.0,38709617.0,E MTAB 13263:NG 32417 R2114 dre C1 clobetasolpropionate lib667286 10169 3 ,0:151 1:151,A:3142106866;C:2677986743;G:2767050957;T:3103028799;N:130969,151,151,,,3142106866,2677986743,2767050957,3103028799,130969,ERX11232844,ERS16254763,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95418,0.95523,0.12548,0.12397,0.66026,0.66016,0.46767,0.47253,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11825,ERR11834688,ERX11232841,ERS16254760,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 2,SAMEA114265802,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265802|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 2|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:0|organism part:whole organism|rin:9.6|sample name:E MTAB 13263:Sample 2|scientific name:Danio rerio|sex:na|stimulus:n1|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 2 p,Sample 2 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2111_dre_nc_clobetasolpropionate_lib673070_10180_1_1.fastq.gz NG-32417_R2111_dre_nc_clobetasolpropionate_lib673070_10180_1_2.fastq.gz,fastq fastq,11827163184.0,39162792.0,E MTAB 13263:NG 32417 R2111 dre nc clobetasolpropionate lib673070 10180 1 ,0:151 1:151,A:3256286612;C:2657918302;G:2701905502;T:3210846546;N:206222,151,151,,,3256286612,2657918302,2701905502,3210846546,206222,ERX11232841,ERS16254760,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95203,0.95292,0.12277,0.11772,0.66048,0.65837,0.47243,0.4729,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11826,ERR11834693,ERX11232846,ERS16254765,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 7,SAMEA114265807,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265807|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 7|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.7|sample name:E MTAB 13263:Sample 7|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 7 p,Sample 7 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2116_dre_nc_imq_clobetasolpropionate_lib667288_10169_3_1.fastq.gz NG-32417_R2116_dre_nc_imq_clobetasolpropionate_lib667288_10169_3_2.fastq.gz,fastq fastq,11114382784.0,36802592.0,E MTAB 13263:NG 32417 R2116 dre nc imq clobetasolpropionate lib667288 10169 3 ,0:151 1:151,A:2997376944;C:2547742011;G:2613273908;T:2955864850;N:125071,151,151,,,2997376944,2547742011,2613273908,2955864850,125071,ERX11232846,ERS16254765,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95392,0.9539,0.12951,0.12707,0.65628,0.65731,0.48132,0.48179,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11827,ERR11834695,ERX11232848,ERS16254767,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 9,SAMEA114265809,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265809|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 9|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.9|sample name:E MTAB 13263:Sample 9|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 9 p,Sample 9 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2118_dre_nc_imq_clobetasolpropionate_lib667290_10169_3_1.fastq.gz NG-32417_R2118_dre_nc_imq_clobetasolpropionate_lib667290_10169_3_2.fastq.gz,fastq fastq,9422294300.0,31199650.0,E MTAB 13263:NG 32417 R2118 dre nc imq clobetasolpropionate lib667290 10169 3 ,0:151 1:151,A:2541445212;C:2157798531;G:2214381344;T:2508560588;N:108625,151,151,,,2541445212,2157798531,2214381344,2508560588,108625,ERX11232848,ERS16254767,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95337,0.95433,0.13046,0.12718,0.65845,0.65815,0.48198,0.48414,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11828,ERR11834687,ERX11232840,ERS16254759,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 12,SAMEA114265801,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265801|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 12|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:9.7|sample name:E MTAB 13263:Sample 12|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 12 p,Sample 12 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2121_dre_C1_imq_clobetasolpropionate_lib667293_10169_3_2.fastq.gz NG-32417_R2121_dre_C1_imq_clobetasolpropionate_lib667293_10169_3_1.fastq.gz,fastq fastq,9285032280.0,30745140.0,E MTAB 13263:NG 32417 R2121 dre C1 imq clobetasolpropionate lib667293 10169 3 ,0:151 1:151,A:2504765552;C:2110325477;G:2196928032;T:2472905759;N:107460,151,151,,,2504765552,2110325477,2196928032,2472905759,107460,ERX11232840,ERS16254759,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95411,0.95451,0.12502,0.12228,0.65829,0.65774,0.48259,0.47946,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11829,ERR11834694,ERX11232847,ERS16254766,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 8,SAMEA114265808,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265808|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 8|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:4000|organism part:whole organism|rin:9.5|sample name:E MTAB 13263:Sample 8|scientific name:Danio rerio|sex:na|stimulus:immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 8 p,Sample 8 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2117_dre_nc_imq_clobetasolpropionate_lib667289_10169_3_1.fastq.gz NG-32417_R2117_dre_nc_imq_clobetasolpropionate_lib667289_10169_3_2.fastq.gz,fastq fastq,10247527118.0,33932209.0,E MTAB 13263:NG 32417 R2117 dre nc imq clobetasolpropionate lib667289 10169 3 ,0:151 1:151,A:2766675319;C:2342307825;G:2405294253;T:2733131648;N:118073,151,151,,,2766675319,2342307825,2405294253,2733131648,118073,ERX11232847,ERS16254766,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.9541,0.95434,0.13364,0.13098,0.6576,0.6565,0.48068,0.48029,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11830,ERR11834692,ERX11232845,ERS16254764,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 6,SAMEA114265806,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265806|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 6|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250|organism part:whole organism|rin:10|sample name:E MTAB 13263:Sample 6|scientific name:Danio rerio|sex:na|stimulus:immunosuppression|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 6 p,Sample 6 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2115_dre_C1_clobetasolpropionate_lib667287_10169_3_1.fastq.gz NG-32417_R2115_dre_C1_clobetasolpropionate_lib667287_10169_3_2.fastq.gz,fastq fastq,10440673634.0,34571767.0,E MTAB 13263:NG 32417 R2115 dre C1 clobetasolpropionate lib667287 10169 3 ,0:151 1:151,A:2805459166;C:2397120759;G:2463616298;T:2774359989;N:117422,151,151,,,2805459166,2397120759,2463616298,2774359989,117422,ERX11232845,ERS16254764,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95568,0.9548,0.13056,0.12563,0.65882,0.66332,0.49162,0.48586,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11831,ERR11834685,ERX11232838,ERS16254757,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 10,SAMEA114265799,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265799|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 10|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:9.4|sample name:E MTAB 13263:Sample 10|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 10 p,Sample 10 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2119_dre_C1_imq_clobetasolpropionate_lib667291_10169_3_2.fastq.gz NG-32417_R2119_dre_C1_imq_clobetasolpropionate_lib667291_10169_3_1.fastq.gz,fastq fastq,11267068246.0,37308173.0,E MTAB 13263:NG 32417 R2119 dre C1 imq clobetasolpropionate lib667291 10169 3 ,0:151 1:151,A:3042432765;C:2574394239;G:2642086000;T:3008026288;N:128954,151,151,,,3042432765,2574394239,2642086000,3008026288,128954,ERX11232838,ERS16254757,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.95345,0.95411,0.13324,0.13062,0.65796,0.65892,0.48176,0.48174,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 11832,ERR11834686,ERX11232839,ERS16254758,ERP150242,PRJEB65099,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E-MTAB-13263,Transcriptome Analysis,Within the regulatory framework on the approval of new substances the assessment of immunotoxic modes of action MoA is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound specific response of zebrafish embryos to two immunomodulative reference substances clobetasol propionate CP CAS 25122 46 7 and imiquimod IMQ CAS 99011 02 6 on the global level of gene expression. The obtained results were used to i identify potential biomarker candidates for the assessment of immunotoxic MoAs ii identify compound specific molecular signatures iii evaluate the reliability of data acquisition using OMICs coupled approaches and iv to assess the suitability of the zebrafish embryo as an alternative model for the human representative investigation of psoriatic effects. For this the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP induced immunosuppression in absence or presence of an IMQ induced immune challenge i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test OECD 236 15 fertilized eggs were exposed to either CP 250 nM or to IMQ 4000 nM or to a combination of both under semi static conditions. Exposure to CP started at 2 hpf until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without xxx nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit Macherey Nagel following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system Agilent before coding RNA was purified PolyA selection with TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina NovaSeq 6000 System Illumina in 150 bp paired end mode producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,,Protocols: At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,Sample 11,SAMEA114265800,"Department Ecotoxicogenomics, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany",ENA FIRST PUBLIC:2023 12 01T00:42:51Z|ENA LAST UPDATE:2023 12 01T00:42:51Z|External Id:SAMEA114265800|INSDC center name:Department Ecotoxicogenomics Fraunhofer Institute for Molecular Biology and Applied Ecology IME Schmallenberg Germany|INSDC first public:2023 12 01T00:42:51Z|INSDC last update:2023 12 01T00:42:51Z|INSDC status:public|Submitter Id:E MTAB 13263:Sample 11|age:3|broker name:ArrayExpress|collection date:not collected|common name:zebrafish|developmental stage:embryo stage|genotype:AB|geographic location country and/or sea:not collected|isolate:not applicable|nominal concentration in medium:250 CP + 4000 IMQ|organism part:whole organism|rin:10|sample name:E MTAB 13263:Sample 11|scientific name:Danio rerio|sex:na|stimulus:immunosuppression and immunostimulation|strain:Wildtype AB,,,,,,,,,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,E MTAB 13263:Sample 11 p,Sample 11 p,Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,At 72 hpf for each sample all 15 zebrafish embryos were picked from the glass well and carefully transferred to a 1.5 ml low binding Eppendorf tube. Excessive liquid was removed and tubes were placed on ice to euthanize the embryos. For detailed information about embryonic incubation conditions and husbandry of adult broodstock please refer to the “Growth protocol”. For detailed information about exposure duration and conditions please refer to the “Treatment protocol”. Adult animal husbandry: All experiments were performed using zebrafish Danio rerio embryos of the wild type strain AB. Adult fish were originally obtained from West Aquarium GmbH Germany and were kept in 150 l tanks under flow through conditions at 26 ± 2 °C and a 12:12 light/dark cycle at the Fraunhofer Institute for molecular biology and applied ecology IME Schmallenberg Germany. TetraMin® Tetra Werke Germany was used as daily main feed regularly supplemented with nauplii of Artemia salina. Fish were constantly bred for several generations without xxx between tanks making the embryos deriving from different spawning groups tanks independent biological replicates. For egg collection glass spawning trays equipped with whirled cords of green glass pearls to stimulate spawning and covered with wired mesh to prevent cannibalism were put into the tanks the day before tests were started OECD 2013. The morning post eggs were collected from the trays using a sieve rinsed with pre tempered system water to remove remaining feces gathered in glass jars filled with pre tempered aerated system water and stored at a controlled temperature of 26 ± 1 °C until further use. Fish embryo incubation: Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. For details on the treatment see “treatment protocol” Clobetasol propionate 21 Chlor 9 fluor 11β 17 dihydroxy 16β methylpregna 1 4 dien 3 20 dion 17 propionat purity ≥ 98 % CAS no. 25122 46 7 and Imiquimod 1 2 methylpropyl 1H imidazo[4 5 c]quinoline 4 amine purity ≥ 95 % CAS no. 9901 02 6 were purchased from BIOMOL GmbH Hamburg Germany. For IMQ a stock solution in acetone with a nominal concentration of 0.1 mg/ml was prepared. For CP a stock solution in dimethyl sulfoxide DMSO with a nominal concentration of 2.5 mM was prepared. To obtain a test solution with a nominal concentration of 4666 nM IMQ 2242.5 µl of the IMQ stock solution was added to a glass bottle. To equalize the amount of added acetone amongst all solutions equal volume of pure acetone without xxx was added to glass bottles for all remaining test solutions. For all test solutions containing 250 nM CP 20 µl of the CP stock solution was added to the glass bottles post complete evaporation of the acetone and filled with copper reduced tap water in the following referred to as cu red water to a final volume of 200 ml. Test solutions without xxx were prepared alike adding 20 µl DMSO without xxx. All solutions were sonicated for 30 min at room temperature Sonorex RK 100H Bandelin Germany. Final solutions were thus cu red water with 0.01 % DMSO 4666 nM IMQ in cu red water with 0.01 % DMSO 250 nM CP in cu red water with 0.01 % DMSO and 4666 nM IMQ plus 250 nM CP in cu red water with 0.01 % DMSO. Due to a mandatory dilution during the methodological procedure a nominal working concentration of 4000 nM was achieved for IMQ in these solutions. The test solutions were freshly prepared one day prior the start of the experiment and stored at room temperature in the dark. They were constantly aerated and used over the course of the 72 hours exposure experiment. The glass wells for the incubation of the embryos were pre saturated with the respective test solution overnight and renewed at the beginning of the experiment. In order to minimize the differences between replicates and conditions in exposure durations the selection of embryos was performed in two steps: at 2 hpf approx. 50 embryos from spawning batches with a minimum fertilization rate ≥ 90 % were transferred to previously overnight saturated glass petri dishes 6 cm diameter refilled with 7 ml of the corresponding test solutions for each replicate and condition. Embryos of the control and the IMQ condition were thus transferred to medium without xxx compound embryos of the CP and combined CP+IMQ condition were transferred to medium supplemented with 250 nM CP. Once in solution 15 healthy and fertilized embryos indicated by a well shaped blastodisc between the 128 cell and 1k cell stage of development were selected per replicate and condition for experiments. Embryos were incubated at a controlled temperature of 26 ± 1°C and a 14:10 h light/dark cycle artificially created with tubular fluorescent lamps with the lights switched on at 8 am. Medium was refreshed at 24 hpf by renewing half its volume. Embryos were inspected microscopically on a daily basis and at the end of the experiment using an OZL 451 stereomicroscope KERN & Sohn Germany in order to monitor the occurrence of indicators of lethality as defined by the OECD FET test guideline namely coagulation lack of somite formation lack of a heartbeat non detachment of the tail OECD 2013 as well as further indicators of malformations such as scoliosis formation of edema or lack of pigmentation. At 47 hpf embryos were manually dechorionated using two pointy forceps. At 48 hpf the immune challenge was started by transferring the embryos of the IMQ condition and the combined CP+IMQ condition to the respective test solution supplemented with IMQ. For this and in order to equalized the subsequent dilution of media between replicates and conditions embryos were gathered in 1 ml of the aged medium and transferred with this volume into newly saturated glass dishes filled with 6 ml of the corresponding fresh medium. Due to this dilution a nominal working concentration of 4000 nM IMQ was achieved for the immune challenge. Embryos of the control condition and the CP condition were handled alike but without xxx the type of test solution. RNA was extracted using a NucleoSpin RNA/Protein kit Macherey Nagel Düren Germany. Briefly frozen embryos were transferred to screw cap tubes filled with 350 µl kit specific lysis buffer RP1 buffer and Lysing Matrix D ceramic beads MP Biomedicals Irvine USA and homogenized using a FastPrep 24 MP Biomedicals Irvine USA device at 5 m/s for 1 min. RP1 buffer was freshly prepared with TCEP as reducing agent prior extraction. Further steps were performed following the manufacturer's extraction protocol. As quality measures purity and concentration of extracted RNA were determined using a NanoDrop 2000 device Thermo Scientific Waltham USA and RNA integrity numbers RIN were determined using a Bioanalyzer 2100 device Agilent Santa Clara USA with samples prepared using a RNA Pico 6000 kit Agilent Santa Clara USA following the manufacturer's instructions. Only samples with a RIN > 9 were used for downstream analysis. Samples were stored at 80°C until they were send to the sequencing facility on dry ice. High quality RNA samples with RIN > 9 were selected for RNA seq library preparation and sequencing performed by Eurofins Genomics Germany GmbH Ebersberg Germany. Briefly Poly A containing protein coding mRNA molecules were purified from total RNA randomly fragmentated and subjected to random primed strand specific cDNA library preparation using the TruSeq RNA library Prep Kit v2 Illumina following the manufacturer's instructions.,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP150242,Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profiling of TLR 7 mediated immune challenge in zebrafish embryos in the presence and absence of glucocorticoid induced immunosuppression,ENA FIRST PUBLIC:2023 12 01|ENA LAST UPDATE:2023 12 01,NG-32417_R2120_dre_C1_imq_clobetasolpropionate_lib667292_10169_3_2.fastq.gz NG-32417_R2120_dre_C1_imq_clobetasolpropionate_lib667292_10169_3_1.fastq.gz,fastq fastq,9922486330.0,32855915.0,E MTAB 13263:NG 32417 R2120 dre C1 imq clobetasolpropionate lib667292 10169 3 ,0:151 1:151,A:2673462020;C:2268840894;G:2345658284;T:2634411815;N:113317,151,151,,,2673462020,2268840894,2345658284,2634411815,113317,ERX11232839,ERS16254758,ERA26742386,European Bioinformatics Institute|European Nucleotide Archive,European Bioinformatics Institute|European Nucleotide Archive,2,0.9551,0.95621,0.1193,0.11675,0.65833,0.65827,0.48596,0.48547,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United Kingdom,2023-12-01,Multi-stage,Multi-stage,Whole Organism,All anatomical structures 13299,ERR984502,ERX1065723,ERS715232,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367580,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367580|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 24 sc 2286404|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCAGCTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 24 sc 2286404|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#24,13741631,Illumina sequencing of library 13741631 constructed from sample accession ERS715232 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TTCAGCTC.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#24.cram,cram,570705070.0,4390039.0,SC RUN 16164 8#24,0:55 1:75,A:167603013;C:88769000;G:138595187;T:175336986;N:400884,55,75,,,167603013,88769000,138595187,175336986,400884,ERX1065723,ERS715232,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.18131,0.44012,0.12372,0.1122,0.96759,0.93835,0.63426,0.57651,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13300,ERR984501,ERX1065722,ERS715231,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367579,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367579|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 23 sc 2286403|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTAGTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 23 sc 2286403|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#23,13741630,Illumina sequencing of library 13741630 constructed from sample accession ERS715231 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TACTAGTC.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#23.cram,cram,682831890.0,5252553.0,SC RUN 16164 8#23,0:55 1:75,A:201267036;C:106752351;G:152982998;T:221347859;N:481646,55,75,,,201267036,106752351,152982998,221347859,481646,ERX1065722,ERS715231,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.19992,0.5315,0.13001,0.13817,0.96025,0.92261,0.60527,0.55787,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13301,ERR984500,ERX1065721,ERS715230,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367578,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367578|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 22 sc 2286402|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGATTC is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 22 sc 2286402|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#22,13741629,Illumina sequencing of library 13741629 constructed from sample accession ERS715230 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCAGATTC.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#22.cram,cram,822730220.0,6328694.0,SC RUN 16164 8#22,0:55 1:75,A:241693794;C:130571512;G:177853465;T:272028770;N:582679,55,75,,,241693794,130571512,177853465,272028770,582679,ERX1065721,ERS715230,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.2212,0.54203,0.15635,0.13659,0.96159,0.9221,0.59031,0.55109,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13302,ERR984499,ERX1065720,ERS715229,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367577,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367577|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 21 sc 2286401|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TATGCCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 21 sc 2286401|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#21,13741628,Illumina sequencing of library 13741628 constructed from sample accession ERS715229 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TATGCCAG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#21.cram,cram,616663190.0,4743563.0,SC RUN 16164 8#21,0:55 1:75,A:176848806;C:99629161;G:137029831;T:202730142;N:425250,55,75,,,176848806,99629161,137029831,202730142,425250,ERX1065720,ERS715229,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.21864,0.57174,0.1516,0.14103,0.95905,0.9192,0.55513,0.51633,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13303,ERR984498,ERX1065719,ERS715228,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367576,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367576|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 20 sc 2286400|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGGCTCAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 20 sc 2286400|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#20,13741627,Illumina sequencing of library 13741627 constructed from sample accession ERS715228 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TGGCTCAG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#20.cram,cram,708059950.0,5446615.0,SC RUN 16164 8#20,0:55 1:75,A:195264246;C:129715499;G:160155422;T:222438911;N:485872,55,75,,,195264246,129715499,160155422,222438911,485872,ERX1065719,ERS715228,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.19642,0.48584,0.11921,0.11811,0.95422,0.91843,0.62494,0.56646,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13304,ERR984497,ERX1065718,ERS715227,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367575,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367575|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 19 sc 2286399|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo wild type for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCATTGAG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 19 sc 2286399|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#19,13741626,Illumina sequencing of library 13741626 constructed from sample accession ERS715227 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCATTGAG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#19.cram,cram,651943890.0,5014953.0,SC RUN 16164 8#19,0:55 1:75,A:191809575;C:111573276;G:144109125;T:204007845;N:444069,55,75,,,191809575,111573276,144109125,204007845,444069,ERX1065718,ERS715227,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.17909,0.44616,0.12326,0.12424,0.96256,0.93432,0.56606,0.52819,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13305,ERR984496,ERX1065717,ERS715226,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367574,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367574|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 18 sc 2286398|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTATGCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 18 sc 2286398|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#18,13741625,Illumina sequencing of library 13741625 constructed from sample accession ERS715226 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TGTATGCG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#18.cram,cram,610476620.0,4695974.0,SC RUN 16164 8#18,0:55 1:75,A:181332724;C:99472616;G:132687929;T:196571397;N:411954,55,75,,,181332724,99472616,132687929,196571397,411954,ERX1065717,ERS715226,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.22636,0.55559,0.16123,0.16819,0.96025,0.92967,0.59576,0.54685,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13306,ERR984495,ERX1065716,ERS715225,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367573,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367573|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 17 sc 2286397|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCAGTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 17 sc 2286397|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#17,13741624,Illumina sequencing of library 13741624 constructed from sample accession ERS715225 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCCAGTCG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#17.cram,cram,712320830.0,5479391.0,SC RUN 16164 8#17,0:55 1:75,A:204316194;C:115198795;G:158608369;T:233717227;N:480245,55,75,,,204316194,115198795,158608369,233717227,480245,ERX1065716,ERS715225,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.2492,0.59739,0.17155,0.14862,0.95657,0.92056,0.58039,0.53475,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13307,ERR984494,ERX1065715,ERS715224,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367572,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367572|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 16 sc 2286396|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAAGTTCG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 16 sc 2286396|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#16,13741623,Illumina sequencing of library 13741623 constructed from sample accession ERS715224 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TAAGTTCG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#16.cram,cram,793769340.0,6105918.0,SC RUN 16164 8#16,0:55 1:75,A:232264866;C:130235228;G:171709756;T:259013732;N:545758,55,75,,,232264866,130235228,171709756,259013732,545758,ERX1065715,ERS715224,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.22065,0.5061,0.15274,0.13242,0.95779,0.92575,0.57619,0.53869,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13308,ERR984493,ERX1065714,ERS715223,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367571,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367571|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 15 sc 2286395|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCAGGAGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 15 sc 2286395|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#15,13741622,Illumina sequencing of library 13741622 constructed from sample accession ERS715223 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCAGGAGG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#15.cram,cram,785479760.0,6042152.0,SC RUN 16164 8#15,0:55 1:75,A:221631721;C:150326010;G:172964474;T:240036379;N:521176,55,75,,,221631721,150326010,172964474,240036379,521176,ERX1065714,ERS715223,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.26761,0.37751,0.19858,0.09301,0.95162,0.93129,0.56282,0.54289,55,75,B,B,mate1-mate2 similar by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13309,ERR984492,ERX1065713,ERS715222,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367570,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367570|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 14 sc 2286394|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCTCACGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 14 sc 2286394|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#14,13741621,Illumina sequencing of library 13741621 constructed from sample accession ERS715222 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCTCACGG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#14.cram,cram,673293530.0,5179181.0,SC RUN 16164 8#14,0:55 1:75,A:188203068;C:115934699;G:155324541;T:213374769;N:456453,55,75,,,188203068,115934699,155324541,213374769,456453,ERX1065713,ERS715222,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.21855,0.57586,0.15197,0.14501,0.95946,0.93077,0.56453,0.52351,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13310,ERR984491,ERX1065712,ERS715221,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367569,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367569|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 13 sc 2286393|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo heterozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACTTCGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 13 sc 2286393|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#13,13741620,Illumina sequencing of library 13741620 constructed from sample accession ERS715221 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TACTTCGG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#13.cram,cram,783634020.0,6027954.0,SC RUN 16164 8#13,0:55 1:75,A:231435775;C:129417634;G:178939153;T:243302897;N:538561,55,75,,,231435775,129417634,178939153,243302897,538561,ERX1065712,ERS715221,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.20287,0.52947,0.14655,0.13479,0.96299,0.93703,0.56376,0.52626,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13311,ERR984490,ERX1065711,ERS715220,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367568,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367568|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 12 sc 2286392|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGAACTGG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 12 sc 2286392|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#12,13741619,Illumina sequencing of library 13741619 constructed from sample accession ERS715220 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TGAACTGG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#12.cram,cram,746397730.0,5741521.0,SC RUN 16164 8#12,0:55 1:75,A:217959769;C:123138989;G:158254272;T:246531930;N:512770,55,75,,,217959769,123138989,158254272,246531930,512770,ERX1065711,ERS715220,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.20695,0.57674,0.14123,0.14978,0.95899,0.9138,0.56705,0.57522,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13312,ERR984489,ERX1065710,ERS715219,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367567,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:01:26Z|External Id:SAMEA3367567|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:01:26Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 11 sc 2286391|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTGGTATG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 11 sc 2286391|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#11,13741618,Illumina sequencing of library 13741618 constructed from sample accession ERS715219 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TTGGTATG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#11.cram,cram,630077890.0,4846753.0,SC RUN 16164 8#11,0:55 1:75,A:181555459;C:107913967;G:131155798;T:209016157;N:436509,55,75,,,181555459,107913967,131155798,209016157,436509,ERX1065710,ERS715219,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.23034,0.58664,0.14994,0.16031,0.9517,0.9134,0.43491,0.55537,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13313,ERR984488,ERX1065709,ERS715218,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367566,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:57Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367566|INSDC center name:SC|INSDC first public:2015 08 18T09:04:57Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 10 sc 2286390|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAACGCTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 10 sc 2286390|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#10,13741617,Illumina sequencing of library 13741617 constructed from sample accession ERS715218 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TAACGCTG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#10.cram,cram,522425410.0,4018657.0,SC RUN 16164 8#10,0:55 1:75,A:136789388;C:88820863;G:131305976;T:165156817;N:352366,55,75,,,136789388,88820863,131305976,165156817,352366,ERX1065709,ERS715218,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.16976,0.61187,0.10677,0.16777,0.96096,0.93093,0.59603,0.58893,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13314,ERR984487,ERX1065708,ERS715217,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367565,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367565|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 9 sc 2286389|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCGAAGTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 9 sc 2286389|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#9,13741616,Illumina sequencing of library 13741616 constructed from sample accession ERS715217 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCGAAGTG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#9.cram,cram,662372100.0,5095170.0,SC RUN 16164 8#9,0:55 1:75,A:186346528;C:114321445;G:137139385;T:224106023;N:458719,55,75,,,186346528,114321445,137139385,224106023,458719,ERX1065708,ERS715217,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.22854,0.63729,0.14734,0.16003,0.95089,0.90751,0.51773,0.50977,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13315,ERR984486,ERX1065707,ERS715216,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367564,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367564|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 8 sc 2286388|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCATTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 8 sc 2286388|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#8,13741615,Illumina sequencing of library 13741615 constructed from sample accession ERS715216 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TTCCATTG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#8.cram,cram,857007060.0,6592362.0,SC RUN 16164 8#8,0:55 1:75,A:257958701;C:131661477;G:190276240;T:276500331;N:610311,55,75,,,257958701,131661477,190276240,276500331,610311,ERX1065707,ERS715216,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.21266,0.595,0.14225,0.14037,0.95964,0.92232,0.54595,0.53111,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13316,ERR984485,ERX1065706,ERS715215,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367563,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367563|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 7 sc 2286387|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTCTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 7 sc 2286387|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#7,13741614,Illumina sequencing of library 13741614 constructed from sample accession ERS715215 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TAGTCTTG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#7.cram,cram,742942590.0,5714943.0,SC RUN 16164 8#7,0:55 1:75,A:228799118;C:117009382;G:166056205;T:230565934;N:511951,55,75,,,228799118,117009382,166056205,230565934,511951,ERX1065706,ERS715215,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.19264,0.50767,0.1428,0.15173,0.9666,0.94606,0.57413,0.53902,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13317,ERR984484,ERX1065705,ERS715214,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367562,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367562|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 6 sc 2286386|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGTGGTTG is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 6 sc 2286386|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#6,13741613,Illumina sequencing of library 13741613 constructed from sample accession ERS715214 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TGTGGTTG.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#6.cram,cram,908951420.0,6991934.0,SC RUN 16164 8#6,0:55 1:75,A:266807632;C:158160652;G:194742499;T:288619707;N:620930,55,75,,,266807632,158160652,194742499,288619707,620930,ERX1065705,ERS715214,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.20021,0.52069,0.13208,0.1197,0.95775,0.93324,0.52946,0.52456,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13318,ERR984483,ERX1065704,ERS715213,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367561,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367561|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 5 sc 2286385|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TCCTCAAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 5 sc 2286385|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#5,13741612,Illumina sequencing of library 13741612 constructed from sample accession ERS715213 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TCCTCAAT.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#5.cram,cram,771659330.0,5935841.0,SC RUN 16164 8#5,0:55 1:75,A:225161011;C:121434126;G:174639826;T:249876341;N:548026,55,75,,,225161011,121434126,174639826,249876341,548026,ERX1065704,ERS715213,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.21069,0.57513,0.14228,0.12501,0.95672,0.92488,0.54397,0.52111,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13319,ERR984482,ERX1065703,ERS715212,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367560,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367560|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 4 sc 2286384|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TACAGGAT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 4 sc 2286384|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#4,13741611,Illumina sequencing of library 13741611 constructed from sample accession ERS715212 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TACAGGAT.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#4.cram,cram,784183790.0,6032183.0,SC RUN 16164 8#4,0:55 1:75,A:219327080;C:128313123;G:169475262;T:266538585;N:529740,55,75,,,219327080,128313123,169475262,266538585,529740,ERX1065703,ERS715212,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.20392,0.58702,0.12804,0.12156,0.96047,0.9133,0.61438,0.53735,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13320,ERR984481,ERX1065702,ERS715211,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367559,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:40:24Z|External Id:SAMEA3367559|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:40:24Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 3 sc 2286383|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TAGTGACT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 3 sc 2286383|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#3,13741610,Illumina sequencing of library 13741610 constructed from sample accession ERS715211 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TAGTGACT.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#3.cram,cram,810265170.0,6232809.0,SC RUN 16164 8#3,0:55 1:75,A:237117705;C:130774361;G:170817877;T:270997878;N:557349,55,75,,,237117705,130774361,170817877,270997878,557349,ERX1065702,ERS715211,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.22414,0.5931,0.14561,0.13304,0.95651,0.91423,0.59465,0.54152,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13321,ERR984480,ERX1065701,ERS715210,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367558,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:55Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367558|INSDC center name:SC|INSDC first public:2015 08 18T09:04:55Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 2 sc 2286382|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TTCCTGCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 2 sc 2286382|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#2,13741609,Illumina sequencing of library 13741609 constructed from sample accession ERS715210 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TTCCTGCT.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#2.cram,cram,698194770.0,5370729.0,SC RUN 16164 8#2,0:55 1:75,A:190784194;C:110138446;G:188418516;T:208362798;N:490816,55,75,,,190784194,110138446,188418516,208362798,490816,ERX1065701,ERS715210,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.17398,0.58306,0.10399,0.12476,0.96372,0.94468,0.64309,0.54721,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 13322,ERR984479,ERX1065700,ERS715209,ERP006132,PRJEB6584,Transcriptome profiling of embryos collected for one or more alleles identified by the zebrafish mutation project,Transcriptome_profiling_of_embryos_collected_for_one_or_more_alleles_identified_by_the_zebrafish_mutation_project-sc-3191,Transcriptome Analysis,Paired end sequence data from the IlluminaHiSeq was prepared for zebrafish embryos collected for one or more alleles identified by the Zebrafish Mutation Project for transcriptome profiling.,,,,,SAMEA3367557,SC,ArrayExpress DevelopmentalStage:Segmentation:26+ somites ZFS:0000028 Pharyngula:Prim 5 ZFS:0000029|ArrayExpress OrganismPart:Whole embryo|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2015 08 18T09:04:56Z|ENA LAST UPDATE:2018 03 09T00:00:47Z|External Id:SAMEA3367557|INSDC center name:SC|INSDC first public:2015 08 18T09:04:56Z|INSDC last update:2018 03 09T00:00:47Z|INSDC status:public|Submitter Id:ZMP phenotype 175 1 1 sc 2286381|common name:zebrafish|sample description:3 prime end enriched mRNA from a single embryo homozygous for pcna allele sa8962 plus ERCC spike mix 1 Ambion. A 8 base indexing sequence TGCGATCT is bases 13 to 20 of read 1 followed by CG and polyT. More information describing the mutant phenotype can be found at the Wellcome Trust Sanger Institute Zebrafish Mutation Project website http://www.sanger.ac.uk/sanger/Zebrafish Zmpsearch/zmp ph175.|sample name:ZMP phenotype 175 1 1 sc 2286381|scientific name:Danio rerio,,,,,,,,,Illumina HiSeq 2000 paired end sequencing,SC EXP 16164 8#1,13741608,Illumina sequencing of library 13741608 constructed from sample accession ERS715209 for study accession ERP006132. This is part of an Illumina multiplexed sequencing run 16164 8. This submission includes reads tagged with the sequence TGCGATCT.,Transcriptome counting qPCR only,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,ERP006132,Illumina HiSeq 2000 paired end sequencing,ENA FIRST PUBLIC:2015 08 18|ENA LAST UPDATE:2018 11 16,16164_8#1.cram,cram,882032060.0,6784862.0,SC RUN 16164 8#1,0:55 1:75,A:258098215;C:144707482;G:194402683;T:284218124;N:605556,55,75,,,258098215,144707482,194402683,284218124,605556,ERX1065700,ERS715209,ERA466416,The Wellcome Trust Sanger Institute|European Nucleotide Archive,Wellcome Sanger Institute,2,0.21585,0.55375,0.14409,0.13461,0.96015,0.9246,0.61791,0.5495,55,75,T,B,mate1 technical by mapping diff,illumina,hiseq_era,3prime,cdna_unspecified,unknown,bulk,unknown,unknown,,United Kingdom,2015-08-18,Multi-stage,Embryo,Whole Organism,All anatomical structures 24967,SRR25557924,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5_S3_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L001_R2_001.fastq.gz,fastq fastq fastq,783833459.0,6171917.0,GSM7688796 r1,0:8 1:28 2:91,A:165863131;C:117191802;G:128146802;T:150173329;N:269383,8,28,91,,165863131,117191802,128146802,150173329,269383,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89127,,0.221,,0.78317,,0.52195,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24968,SRR25557925,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-3_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L002_R2_001.fastq.gz,fastq fastq fastq,7208342327.0,56758601.0,GSM7688796 r10,0:8 1:28 2:91,A:1518008610;C:1083228714;G:1190089577;T:1369772782;N:3933008,8,28,91,,1518008610,1083228714,1190089577,1369772782,3933008,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.88938,,0.21991,,0.79088,,0.53044,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24969,SRR25557926,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-3_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L003_R2_001.fastq.gz,fastq fastq fastq,7337435414.0,57775082.0,GSM7688796 r11,0:8 1:28 2:91,A:1545051667;C:1103394058;G:1211577972;T:1395133564;N:2375201,8,28,91,,1545051667,1103394058,1211577972,1395133564,2375201,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.88966,,0.2211,,0.78961,,0.53156,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24970,SRR25557927,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-3_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L004_R2_001.fastq.gz,fastq fastq fastq,7218240453.0,56836539.0,GSM7688796 r12,0:8 1:28 2:91,A:1521442237;C:1084242714;G:1191469584;T:1372277539;N:2692975,8,28,91,,1521442237,1084242714,1191469584,1372277539,2692975,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.8888,,0.2174,,0.79172,,0.53311,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24971,SRR25557928,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5_S3_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L002_R2_001.fastq.gz,fastq fastq fastq,774302109.0,6096867.0,GSM7688796 r2,0:8 1:28 2:91,A:163989105;C:115793536;G:126358958;T:148430473;N:242825,8,28,91,,163989105,115793536,126358958,148430473,242825,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89371,,0.2215,,0.78253,,0.53282,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24972,SRR25557929,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5_S3_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L003_R2_001.fastq.gz,fastq fastq fastq,790430093.0,6223859.0,GSM7688796 r3,0:8 1:28 2:91,A:167465790;C:118269595;G:129024496;T:151447225;N:164063,8,28,91,,167465790,118269595,129024496,151447225,164063,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89284,,0.21955,,0.78356,,0.53369,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24973,SRR25557930,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5_S3_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5_S3_L004_R2_001.fastq.gz,fastq fastq fastq,778936974.0,6133362.0,GSM7688796 r4,0:8 1:28 2:91,A:165013558;C:116498474;G:127146290;T:149314806;N:162814,8,28,91,,165013558,116498474,127146290,149314806,162814,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89195,,0.2209,,0.78196,,0.53121,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24974,SRR25557931,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-2_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L001_R2_001.fastq.gz,fastq fastq fastq,4174244890.0,32868070.0,GSM7688796 r5,0:8 1:28 2:91,A:882060228;C:627098568;G:684890393;T:795503862;N:1441319,8,28,91,,882060228,627098568,684890393,795503862,1441319,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89137,,0.22041,,0.77926,,0.52991,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24975,SRR25557932,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-2_S2_L002_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L002_R2_001.fastq.gz,fastq fastq fastq,4150210902.0,32678826.0,GSM7688796 r6,0:8 1:28 2:91,A:878218072;C:623662333;G:679977758;T:790589138;N:1325865,8,28,91,,878218072,623662333,679977758,790589138,1325865,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.89264,,0.22159,,0.78121,,0.52989,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24976,SRR25557933,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-2_S2_L003_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L003_R2_001.fastq.gz,fastq fastq fastq,4198043547.0,33055461.0,GSM7688796 r7,0:8 1:28 2:91,A:888287475;C:630914324;G:688006958;T:799794077;N:1044117,8,28,91,,888287475,630914324,688006958,799794077,1044117,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.8912,,0.21889,,0.7822,,0.52774,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24977,SRR25557934,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-2_S2_L004_I1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R1_001.fastq.gz MUTSU2_AllGFP-S5-2_S2_L004_R2_001.fastq.gz,fastq fastq fastq,4154784553.0,32714839.0,GSM7688796 r8,0:8 1:28 2:91,A:880520885;C:624179045;G:680076172;T:791297938;N:976309,8,28,91,,880520885,624179045,680076172,791297938,976309,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.8927,,0.22063,,0.78216,,0.5346,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24978,SRR25557935,SRX21286763,SRS18536876,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 AllGFP S5,GSM7688796,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 AllGFP S5,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688796,GSM7688796: MUTSU2 AllGFP S5; Danio rerio; RNA Seq,GSM7688796 r1,GSM7688796,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_AllGFP-S5-3_S2_L001_I1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R1_001.fastq.gz MUTSU2_AllGFP-S5-3_S2_L001_R2_001.fastq.gz,fastq fastq fastq,7231973725.0,56944675.0,GSM7688796 r9,0:8 1:28 2:91,A:1522035629;C:1085783001;G:1195535350;T:1374885853;N:3725592,8,28,91,,1522035629,1085783001,1195535350,1374885853,3725592,SRX21286763,SRS18536876,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.88833,,0.22076,,0.79056,,0.52744,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24979,SRR25557936,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8_S2_L001_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L001_R2_001.fastq.gz,fastq fastq fastq,699161670.0,5505210.0,GSM7688795 r1,0:8 1:28 2:91,A:149927051;C:100829317;G:116075103;T:133908276;N:234363,8,28,91,,149927051,100829317,116075103,133908276,234363,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85081,,0.21257,,0.80426,,0.52779,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24980,SRR25557937,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-3_S1_L002_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L002_R2_001.fastq.gz,fastq fastq fastq,8174836330.0,64368790.0,GSM7688795 r10,0:8 1:28 2:91,A:1748532358;C:1187854561;G:1356279755;T:1560491610;N:4401606,8,28,91,,1748532358,1187854561,1356279755,1560491610,4401606,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85069,,0.20989,,0.81049,,0.52069,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24981,SRR25557938,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-3_S1_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L003_R2_001.fastq.gz,fastq fastq fastq,8307872640.0,65416320.0,GSM7688795 r11,0:8 1:28 2:91,A:1778112295;C:1207569588;G:1377139686;T:1587359299;N:2704252,8,28,91,,1778112295,1207569588,1377139686,1587359299,2704252,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85151,,0.21054,,0.80813,,0.52675,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24982,SRR25557939,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-3_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L004_I1_001.fastq.gz,fastq fastq fastq,8197523483.0,64547429.0,GSM7688795 r12,0:8 1:28 2:91,A:1755321680;C:1190415496;G:1359502433;T:1565500310;N:3076120,8,28,91,,1755321680,1190415496,1359502433,1565500310,3076120,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.84956,,0.20968,,0.81113,,0.52382,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24983,SRR25557940,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8_S2_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L002_I1_001.fastq.gz,fastq fastq fastq,690198899.0,5434637.0,GSM7688795 r2,0:8 1:28 2:91,A:147896108;C:99606794;G:114681063;T:132140792;N:227210,8,28,91,,147896108,99606794,114681063,132140792,227210,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85228,,0.21261,,0.803,,0.52382,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24984,SRR25557941,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8_S2_L003_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L003_R2_001.fastq.gz,fastq fastq fastq,702239515.0,5529445.0,GSM7688795 r3,0:8 1:28 2:91,A:150477574;C:101266550;G:116878466;T:134415299;N:141606,8,28,91,,150477574,101266550,116878466,134415299,141606,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85364,,0.21344,,0.80346,,0.52158,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24985,SRR25557942,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8_S2_L004_I1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8_S2_L004_R2_001.fastq.gz,fastq fastq fastq,694537346.0,5468798.0,GSM7688795 r4,0:8 1:28 2:91,A:148876263;C:100202441;G:115455212;T:132982029;N:144673,8,28,91,,148876263,100202441,115455212,132982029,144673,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85238,,0.2138,,0.80379,,0.52691,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24986,SRR25557943,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-2_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L001_I1_001.fastq.gz,fastq fastq fastq,4324997954.0,34055102.0,GSM7688795 r5,0:8 1:28 2:91,A:924471384;C:626141120;G:722856562;T:824050239;N:1494977,8,28,91,,924471384,626141120,722856562,824050239,1494977,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85083,,0.21122,,0.80472,,0.524,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24987,SRR25557944,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-2_S1_L002_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L002_I1_001.fastq.gz,fastq fastq fastq,4306232942.0,33907346.0,GSM7688795 r6,0:8 1:28 2:91,A:920615353;C:623090513;G:721201055;T:819291149;N:1370416,8,28,91,,920615353,623090513,721201055,819291149,1370416,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85335,,0.21315,,0.80206,,0.51508,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24988,SRR25557945,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-2_S1_L003_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L003_I1_001.fastq.gz,fastq fastq fastq,4348436312.0,34239656.0,GSM7688795 r7,0:8 1:28 2:91,A:930204208;C:629005094;G:727756813;T:827747478;N:1095103,8,28,91,,930204208,629005094,727756813,827747478,1095103,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85369,,0.21216,,0.80503,,0.52219,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24989,SRR25557946,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-2_S1_L004_R2_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_R1_001.fastq.gz MUTSU2_OtherGFP-G8-2_S1_L004_I1_001.fastq.gz,fastq fastq fastq,4313915172.0,33967836.0,GSM7688795 r8,0:8 1:28 2:91,A:922742754;C:623127831;G:724345885;T:819819593;N:1037013,8,28,91,,922742754,623127831,724345885,819819593,1037013,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.85456,,0.21236,,0.80223,,0.5216,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 24990,SRR25557947,SRX21286762,SRS18536875,SRP453891,PRJNA1003032,Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in These Cells. [scRNA Seq],GSE240239,Transcriptome Analysis,Background: V0v spinal interneurons are highly conserved glutamatergic commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks we FAC sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression profiled them using microarrays and scRNA seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 mutants and wild type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that by this stage of development evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly we show that Hmx2/3a repress dI2 interneuronal expression of skor1a and nefma two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of V0v interneurons. Our data further suggest that in the absence of both Evx1 and Evx2 V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then later start to express markers of distinct types of inhibitory spinal interneurons or motoneurons. Taken together our findings significantly increase our knowledge of V0v spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type. Overall design: Two samples were analysed in total both at 48 hpf. These two samples represent two biological replicates of all V0v spinal interneurons isolated from embryos from an incross of heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents.,parent bioproject:PRJNA1003022,pubmed:38017520,,MUTSU2 OtherGFP G8,GSM7688795,,source name:Spinal Cord|tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1|geo loc name:missing|collection date:missing,MUTSU2 OtherGFP G8,We performed demultiplexing and counts analysis as per the manufacturer’s instructions using Cell Ranger v4.0.0 software https://www.10xgenomics.com We analyzed the data using Partek Flow Genomic Analysis Software https://www.partek.com/partek flow/. Multiplets were removed by filtering out cells with >12 000 counts and >2 500 detected genes. Sick and/or “leaky” cells were removed by filtering out cells with <500 detected genes and >6% mitochondrial transcripts. We normalized the data using a counts per million CPM algorithm and applied a logarithmic transformation to improve data visualization. The outcome of normalisation was assessed by principal components analysis PCA graph based clustering and Uniform Manifold Approximation and Projection UMAP plotting using the NN Descent method of nearest neighbour type calculation and Euclidean distance metrics. We manually inspected UMAP plots to assess clustering quality based on expression of known V0v spinal interneuron markers. We then fine tuned the clustering by manually deducing and extrapolating cell fate assignments by comparing expression profiles of 48 hpf single cell clusters with the molecular phenotypes of V0v spinal interneurons in 24 hpf and 30 hpf wild type evx1i232 and evx2sa140 single mutant and evx1i232;evx2sa140 double mutant embryos. To perform differential expression analysis in Partek Flow we used the statistically robust Hurdle Model two part model with default parameters. We initially analyzed the two libraries separately. We then combined the data from these two libraries using the Counts Aggregation pipeline in Cell Ranger v4.0.0 and reanalyzed the data as described above. Assembly: Lawson Lab zebrafish transcriptome V4.3.2 https://www.umassmed.edu/lawson lab/reagents/zebrafish transcriptome/ Supplementary files format and content: Tab separated and matrix files.,Spinal Cord,,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz’s L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco’s Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco’s Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell 3’ GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,Embryos obtained from crossing heterozygous evx1i232/+;evx2sa140/+;Tgevx1:EGFPSU2 parents were raised at 28.5oC until they reached the desired developmental stage of 48 hpf as confirmed by analysis of morphological criteria including head trunk angle head and eye size.,tissue:Spinal Cord|cell line:Tgevx1:EGFPSU2|cell type:V0v spinal interneurons|genotype:evx1i232/+;evx2sa140/+ incross|treatment:N1,GSM7688795,GSM7688795: MUTSU2 OtherGFP G8; Danio rerio; RNA Seq,GSM7688795 r1,GSM7688795,1,Embryos were screened for fluorescence from 30 hpf onwards using a fluorescent dissecting microscope. Only EGFP positive embryos were used for dissections and FACS at 48 hpf. Embryos were deyolked dissected and dissociated as described in GSE145916 with the following modifications: Trunk tissue was dissected anteriorly at the boundary between the hindbrain and spinal cord and posteriorly immediately above the end of the yolk extension. To ensure complete dissociation of trunk tissue with the Papain Dissociation System Worthington Biochemical Corporation LK003150 trunks were incubated in 1 ml Papain/DNase mix with gentle rocking at 28.5oC for 40 minutes. The digested tissue was then allowed to settle for 10 seconds before the Papain/DNase mix was carefully decanted until approximately 500 µl remained. Immediately post homogenising the digested tissue mixture with a sterile p200 tip we passed each sample through a 40 µm Flowmi cell strainer Merck BAH136800040 into a sterile microcentrifuge tube. post Papain inactivation samples were resuspended in 1 ml Leibovitz's L 15 medium ThermoFisher Scientific 21083027 + 0.5% FBS and stored on ice. Immediately before FACS DAPI Merck D9542 and Draq5 BioLegend 424101 were added at a final concentration of 5 µg/ml and 5 µM respectively. FACS was performed using a Becton Dickinson FACS Aria III Cell Sorter at the SUNY Upstate Medical University Research Flow Core using the parameters described by Cerda et al. 2008 with the following modifications. Ice cold samples were filtered through 35 µm mesh strainers in to 5 ml round bottomed polystyrene tubes Corning Falcon 352235. All FAC sorting and collection steps were performed at +4oC using a 100 µm nozzle and 20 psi sort pressure. Successive doublet exclusion gates forward scatter height x forward scatter width followed by side scatter height x side scatter width were used to finesse capture of real single cells. Accurate live/dead filtering was performed by selecting for DAPI negative sick cells are DAPI permeant and excluded and Draq 5 positive only healthy nuclei are Draq 5 permeant cells. EGFP positive cells were sorted and fixed using a methanol fixation protocol modified from the 10x Genomics Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com. EGFP positive cells were sorted directly in to 5 ml round bottomed tubes containing 3.5 mls of freshly made pre chilled 90% methanol for HPLC >99% Merck 34860/10% Dulbecco's Phosphate Buffered Saline DPBS No calcium No magnesium Merck D8537 fixative. A tube of EGFP negative cells was also collected to assess fixation efficiency. Sorted cells were incubated on ice for 1 hour before assessing fixation efficiency of the EGFP negative control tube using Trypan Blue ThermoFisher Scientific 15250061 and a hemocytometer. Samples with intact fully fixed cells containing little or no cell debris were stored at +4oC for up to six days prior to rehydrating and performing single cell capture with the 10x Genomics Chromium system please see below. To rehydrate our fixed EGFP positive 48 hpf evx1i232;evx2sa140;Tgevx1:EGFPSU2 cells we first centrifuged each sample at 300 rcf for 10 minutes at +4oC using a swing bucket centrifuge. Next we carefully removed the majority of the supernatant with a sterile p1000 tip until approximately 100 µl remained in the tube. Samples were kept on ice at all times. Each cell pellet was then gently resuspended by adding 2 ml of freshly made pre chilled Rehydration Buffer 1x Dulbecco's Phosphate Buffered Saline no calcium no magnesium Merck D8537 1.0% UltraPure BSA ThermoFisher Scientific AM2616 0.5 u/µl Roche Protector RNase Inhibitor Merck 3335402001 and gently pipetting 10 times. It is important to avoid making foam. We repeated the centrifugation and resuspension in Rehydration Buffer steps as previously. post the second Rehydration step we again centrifuged at 300 rcf for 10 minutes at +4oC before carefully removing all but 30 40 µl of supernatant. Using a sterile p200 tip we carefully resuspended the cell pellet and immediately measured the cell concentration in triplicate using a Bio Rad TC20 automated cell counter Bio Rad 1450102. We also checked a small aliquot under a conventional microscope to ensure we had single cell suspensions. As described by 10x Genomics in their Sample Preparation Demonstrated Protocol “Methanol Fixation of Cells for Single Cell RNA Sequencing” https://www.10xgenomics.com we too recovered approximately 50% of the sorted cells post rehydration. We isolated single cells using a 10x Genomics Chromium system aiming for capture of 10 000 cells per well Chromium Next GEM Chip G Single Cell Kit 1000127. We prepared libraries using a 10x Genomics Chromium Next GEM Single Cell three prime GEM Library and Gel Bead Kit v3.1 10x Genomics 1000128 and sequenced them on an Illumina NextSeq500 to a depth of at least 50 000 reads per cell Illumina NextSeq 500/500 High Output Kit v2.5 150 cycles 20024907.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP453891,,loader:fastq load.py,MUTSU2_OtherGFP-G8-3_S1_L001_R2_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_R1_001.fastq.gz MUTSU2_OtherGFP-G8-3_S1_L001_I1_001.fastq.gz,fastq fastq fastq,8184961024.0,64448512.0,GSM7688795 r9,0:8 1:28 2:91,A:1748819280;C:1187802244;G:1360994184;T:1562975470;N:4223414,8,28,91,,1748819280,1187802244,1360994184,1562975470,4223414,SRX21286762,SRS18536875,SRA1688438,"Lewis Lab, Biology, Syracuse University","Lewis Lab, Biology, Syracuse University",1,0.8493,,0.21072,,0.80967,,0.52593,,91,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-08-07,Multi-stage,Embryo,Spinal Cord,Nervous System 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