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run_metadata

335 rows where devstage_curation_coarse = "Multi-stage" and tissue_curation = "Embryo Imprecise"

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experiment.library_selection 4

technology 3

experiment.platform 3

experiment.library_layout 2

tissue_curation_coarse 1

tissue_curation 1

  • Embryo Imprecise · 335 ✖

experiment.library_strategy 1

experiment.library_source 1

devstage_curation_coarse 1

  • Multi-stage · 335 ✖

devstage_curation 1

Link 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
9892 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 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 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 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 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 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
33722 33722 SRR30640695 SRX26062515 SRS22631699 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         WT24hpfcontrol1   breed:natural spawning of AB adults|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:WT24hpfcontrol1 embryo|BioSampleModel:Model organism or animal                 RNA seq of Wild type zebrafish : 12 embryos in total 2 3mg WT24hpfcontrol1 R WT24hpfcontrol1 R Wild type zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     WT24hpfcontrol1_R1.clean.fq.gz WT24hpfcontrol1_R2.clean.fq.gz fastq fastq 6761688309.0 27496581.0 WT24hpfcontrol1 R1.clean.fq.gz 0:122.99 1:122.92 A:1946719560;C:1442103403;G:1451347536;T:1921456727;N:61083 122 122     1946719560 1442103403 1451347536 1921456727 61083 SRX26062515 SRS22631699 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33723 33723 SRR30640696 SRX26062514 SRS22631698 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1MOMutant3   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1MOMutant3 embryo|BioSampleModel:Model organism or animal                 RNA seq of MO morphants zebrafish : 12 embryos in total 2 5mg PCM1MOMutant3 R PCM1MOMutant3 R MO morphants zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1MOMutant3_R1.clean.fq.gz PCM1MOMutant3_R2.clean.fq.gz fastq fastq 6356420208.0 23407180.0 PCM1MOMutant3 R1.clean.fq.gz 0:135.78 1:135.78 A:1742323782;C:1448163439;G:1451783264;T:1714090084;N:59639 135 135     1742323782 1448163439 1451783264 1714090084 59639 SRX26062514 SRS22631698 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33724 33724 SRR30640697 SRX26062513 SRS22631697 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1MOMutant2   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1MOMutant2 embryo|BioSampleModel:Model organism or animal                 RNA seq of MO morphants zebrafish : 12 embryos in total 2 4mg PCM1MOMutant2 R PCM1MOMutant2 R MO morphants zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1MOMutant2_R1.clean.fq.gz PCM1MOMutant2_R2.clean.fq.gz fastq fastq 6146772469.0 22703012.0 PCM1MOMutant2 R1.clean.fq.gz 0:135.37 1:135.37 A:1647298308;C:1435201745;G:1438871965;T:1625343182;N:57269 135 135     1647298308 1435201745 1438871965 1625343182 57269 SRX26062513 SRS22631697 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33725 33725 SRR30640698 SRX26062512 SRS22631696 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1MOMutant1   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1MOMutant1 embryo|BioSampleModel:Model organism or animal                 RNA seq of MO morphants zebrafish : 12 embryos in total 2 3mg PCM1MOMutant1 R PCM1MOMutant1 R MO morphants zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1MOMutant1_R1.clean.fq.gz PCM1MOMutant1_R2.clean.fq.gz fastq fastq 5701775379.0 21587297.0 PCM1MOMutant1 R1.clean.fq.gz 0:132.06 1:132.06 A:1578153876;C:1282130829;G:1284978375;T:1556460380;N:51919 132 132     1578153876 1282130829 1284978375 1556460380 51919 SRX26062512 SRS22631696 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33726 33726 SRR30640699 SRX26062511 SRS22631695 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOmutant3   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOmutant3 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO mutant zebrafish : 12 embryos in total 2 5mg PCM1KOmutant3 R PCM1KOmutant3 R pcm1 KO mutant zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOmutant3_R1.clean.fq.gz PCM1KOmutant3_R2.clean.fq.gz fastq fastq 4673179612.0 17529406.0 PCM1KOmutant3 R1.clean.fq.gz 0:133.29 1:133.30 A:1266929858;C:1078407007;G:1080490713;T:1247307342;N:44692 133 133     1266929858 1078407007 1080490713 1247307342 44692 SRX26062511 SRS22631695 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33727 33727 SRR30640700 SRX26062510 SRS22631694 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOmutant2   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOmutant2 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO mutant zebrafish : 12 embryos in total 2 4mg PCM1KOmutant2 R PCM1KOmutant2 R pcm1 KO mutant zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOmutant2_R1.clean.fq.gz PCM1KOmutant2_R2.clean.fq.gz fastq fastq 6073820902.0 23767661.0 PCM1KOmutant2 R1.clean.fq.gz 0:127.78 1:127.77 A:1691243395;C:1356197711;G:1357868249;T:1668456939;N:54608 127 127     1691243395 1356197711 1357868249 1668456939 54608 SRX26062510 SRS22631694 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33728 33728 SRR30640701 SRX26062509 SRS22631693 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOmutant1   breed:Tg [ef1:Myr Tdtomato]|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOmutant1 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO mutant zebrafish : 12 embryos in total 2 3mg PCM1KOmutant1 R PCM1KOmutant1 R KO mutant zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOmutant1_R1.clean.fq.gz PCM1KOmutant1_R2.clean.fq.gz fastq fastq 6040589500.0 23826027.0 PCM1KOmutant1 R1.clean.fq.gz 0:126.76 1:126.76 A:1733588631;C:1297359473;G:1301151829;T:1708434373;N:55194 126 126     1733588631 1297359473 1301151829 1708434373 55194 SRX26062509 SRS22631693 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33729 33729 SRR30640702 SRX26062508 SRS22631692 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOcompensate3   breed:Tg [HuC:GFP|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOcompensate3 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO compensate zebrafish : 12 embryos in total 2 5mg PCM1KOcompensate3 R PCM1KOcompensate3 R pcm1 KO compensate zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOcompensate3_R1.clean.fq.gz PCM1KOcompensate3_R2.clean.fq.gz fastq fastq 5632945620.0 21351897.0 PCM1KOcompensate3 R1.clean.fq.gz 0:131.92 1:131.90 A:1561604697;C:1264380828;G:1267746178;T:1539161674;N:52243 131 131     1561604697 1264380828 1267746178 1539161674 52243 SRX26062508 SRS22631692 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33730 33730 SRR30640703 SRX26062507 SRS22631691 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         WT24hpfcontrol3   breed:natural spawning of AB adults|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:WT24hpfcontrol3 embryo|BioSampleModel:Model organism or animal                 RNA seq of Wild type zebrafish : 12 embryos in total 2 5mg WT24hpfcontrol3 R WT24hpfcontrol3 R Wild type zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     WT24hpfcontrol3_R1.clean.fq.gz WT24hpfcontrol3_R2.clean.fq.gz fastq fastq 5047867453.0 19837417.0 WT24hpfcontrol3 R1.clean.fq.gz 0:127.23 1:127.23 A:1412463929;C:1119657233;G:1121642146;T:1394058438;N:45707 127 127     1412463929 1119657233 1121642146 1394058438 45707 SRX26062507 SRS22631691 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33731 33731 SRR30640704 SRX26062506 SRS22631690 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         WT24hpfcontrol2   breed:natural spawning of AB adults|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:WT24hpfcontrol2 embryo|BioSampleModel:Model organism or animal                 RNA seq of Wild type zebrafish : 12 embryos in total 2 4mg WT24hpfcontrol2 R WT24hpfcontrol2 R Wild type zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     WT24hpfcontrol2_R1.clean.fq.gz WT24hpfcontrol2_R2.clean.fq.gz fastq fastq 5408273952.0 21601107.0 WT24hpfcontrol2 R1.clean.fq.gz 0:125.19 1:125.18 A:1510365712;C:1201646307;G:1204419555;T:1491793767;N:48611 125 125     1510365712 1201646307 1204419555 1491793767 48611 SRX26062506 SRS22631690 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33732 33732 SRR30640705 SRX26062505 SRS22631689 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOcompensate2   breed:Tg [HuC:GFP|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOcompensate2 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO compensate zebrafish : 12 embryos in total 2 4mg PCM1KOcompensate2 R PCM1KOcompensate2 R pcm1 KO compensate zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOcompensate2_R1.clean.fq.gz PCM1KOcompensate2_R2.clean.fq.gz fastq fastq 5996379188.0 22765993.0 PCM1KOcompensate2 R1.clean.fq.gz 0:131.70 1:131.70 A:1669800521;C:1339211911;G:1342418096;T:1644890999;N:57661 131 131     1669800521 1339211911 1342418096 1644890999 57661 SRX26062505 SRS22631689 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
33733 33733 SRR30640706 SRX26062504 SRS22631688 SRP532179 PRJNA1159911 Peri Centriolar Material 1 regulates polarized endosome dynamics and neural progenitor fate PRJNA1159911 Other RNA seq was performed on control and maternal zygotic developmentally defective pcm1 morphants MO and pcm1 knock out KO zebrafish embryos at 24 hpf.         PCM1KOcompensate1   breed:Tg [HuC:GFP|age:24hpf|dev stage:adult|collection date:2021 03 19|geo loc name:USA:San Francisco|sex:not determined|tissue:PCM1KOcompensate1 embryo|BioSampleModel:Model organism or animal                 RNA seq of pcm1 KO compensate zebrafish : 12 embryos in total 2 3mg PCM1KOcompensate1 R PCM1KOcompensate1 R pcm1 KO compensate zebrafish     RNA-Seq TRANSCRIPTOMIC PCR PAIRED ILLUMINA Illumina HiSeq 1000   SRP532179     PCM1KOcompensate1_R1.clean.fq.gz PCM1KOcompensate1_R2.clean.fq.gz fastq fastq 5594219881.0 21191291.0 PCM1KOcompensate1 R1.clean.fq.gz 0:131.99 1:131.99 A:1594003450;C:1214578517;G:1215705001;T:1569880851;N:52062 131 131     1594003450 1214578517 1215705001 1569880851 52062 SRX26062504 SRS22631688 SRA1970017 Huazhong Agricultural University, China|Department of Neurosurgery Huazhong Agricultural University, China                       B B biological fallback assumption illumina hiseq_era unknown random_priming unknown bulk unknown unknown   China 2024-09-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37110 37110 SRR942772 SRX327595 SRS463967 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ M. marinum infected GSM1195173   source name:wild type siblings myd88+/+ M.marinum infected 4 dpi|sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo myd88+/+ M. marinum infected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ M.marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo GSM1195173 GSM1195173: myd88+/+ M. marinum infected; Danio rerio; RNA Seq GSM1195173   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195173 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     22_GTCCGC_L005_R2_001_run1.fastq.gz 22_GTCCGC_L005_R1_001_run1.fastq.gz fastq fastq 165821706.0 1625703.0 GSM1195173 r1 0:51 1:51 A:44225689;C:39249878;G:38333339;T:43822905;N:189895 51 51     44225689 39249878 38333339 43822905 189895 SRX327595 SRS463967 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.9274 0.92148 0.11638 0.12031 0.67576 0.66981 0.4779 0.46526 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37111 37111 SRR942773 SRX327595 SRS463967 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ M. marinum infected GSM1195173   source name:wild type siblings myd88+/+ M.marinum infected 4 dpi|sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo myd88+/+ M. marinum infected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ M.marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo GSM1195173 GSM1195173: myd88+/+ M. marinum infected; Danio rerio; RNA Seq GSM1195173   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195173 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     22_GTCCGC_L004_R2_001_run2.fastq.gz 22_GTCCGC_L004_R1_001_run2.fastq.gz fastq fastq 180345690.0 1768095.0 GSM1195173 r2 0:51 1:51 A:47940254;C:42999578;G:41637439;T:47613176;N:155243 51 51     47940254 42999578 41637439 47613176 155243 SRX327595 SRS463967 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.90469 0.90439 0.11306 0.11526 0.67582 0.67361 0.47813 0.47079 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37112 37112 SRR942774 SRX327595 SRS463967 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ M. marinum infected GSM1195173   source name:wild type siblings myd88+/+ M.marinum infected 4 dpi|sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo myd88+/+ M. marinum infected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ M.marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:wild type|tissue:embryo GSM1195173 GSM1195173: myd88+/+ M. marinum infected; Danio rerio; RNA Seq GSM1195173   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195173 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     22_GTCCGC_L008_R2_001_run3.fastq.gz 22_GTCCGC_L008_R1_001_run3.fastq.gz fastq fastq 1897044858.0 18598479.0 GSM1195173 r3 0:51 1:51 A:504429880;C:449611276;G:440496468;T:501740862;N:766372 51 51     504429880 449611276 440496468 501740862 766372 SRX327595 SRS463967 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.92986 0.92294 0.11799 0.119 0.67302 0.67444 0.47284 0.47683 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37113 37113 SRR942769 SRX327594 SRS463966 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ uninfected GSM1195172   source name:wild type siblings myd88+/+ not infected|sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo myd88+/+ uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo GSM1195172 GSM1195172: myd88+/+ uninfected; Danio rerio; RNA Seq GSM1195172   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195172 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     21_CCGTCC_L005_R1_001_run1.fastq.gz 21_CCGTCC_L005_R2_001_run1.fastq.gz fastq fastq 363508110.0 3563805.0 GSM1195172 r1 0:51 1:51 A:98020551;C:85099194;G:82360304;T:97581707;N:446354 51 51     98020551 85099194 82360304 97581707 446354 SRX327594 SRS463966 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91855 0.9152 0.12282 0.12552 0.67464 0.67235 0.46632 0.471 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37114 37114 SRR942770 SRX327594 SRS463966 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ uninfected GSM1195172   source name:wild type siblings myd88+/+ not infected|sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo myd88+/+ uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo GSM1195172 GSM1195172: myd88+/+ uninfected; Danio rerio; RNA Seq GSM1195172   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195172 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     21_CCGTCC_L004_R2_001_run2.fastq.gz 21_CCGTCC_L004_R1_001_run2.fastq.gz fastq fastq 245414754.0 2406027.0 GSM1195172 r2 0:51 1:51 A:66015157;C:57589840;G:55531209;T:66061241;N:217307 51 51     66015157 57589840 55531209 66061241 217307 SRX327594 SRS463966 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.90891 0.90901 0.12166 0.12329 0.67308 0.67243 0.47203 0.46681 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37115 37115 SRR942771 SRX327594 SRS463966 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88+/+ uninfected GSM1195172   source name:wild type siblings myd88+/+ not infected|sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo myd88+/+ uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample wild type siblings myd88+/+ not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:wild type zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:wild type|tissue:embryo GSM1195172 GSM1195172: myd88+/+ uninfected; Danio rerio; RNA Seq GSM1195172   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195172 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     21_CCGTCC_L008_R1_001_run3.fastq.gz 21_CCGTCC_L008_R2_001_run3.fastq.gz fastq fastq 2245421166.0 22013933.0 GSM1195172 r3 0:51 1:51 A:604635890;C:525465932;G:510494585;T:603918129;N:906630 51 51     604635890 525465932 510494585 603918129 906630 SRX327594 SRS463966 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.92278 0.91517 0.12499 0.12518 0.6734 0.67519 0.47029 0.47475 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37116 37116 SRR942767 SRX327593 SRS463965 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88 / M. marinum infected GSM1195171   source name:myd88 mutant myd88 / M.marinum infected 4 dpi|sample type:myd88 mutant zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:myd88 mutant|tissue:embryo myd88 / M. marinum infected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample myd88 mutant myd88 / M.marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:myd88 mutant zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:myd88 mutant|tissue:embryo GSM1195171 GSM1195171: myd88 / M. marinum infected; Danio rerio; RNA Seq GSM1195171   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195171 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     20_ATGTCA_L005_R1_001_run1.fastq.gz 20_ATGTCA_L005_R2_001_run1.fastq.gz fastq fastq 309246150.0 3031825.0 GSM1195171 r1 0:51 1:51 A:83463181;C:72472917;G:70723331;T:82209194;N:377527 51 51     83463181 72472917 70723331 82209194 377527 SRX327593 SRS463965 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91332 0.90654 0.11492 0.11711 0.67326 0.66914 0.47338 0.46933 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37117 37117 SRR942768 SRX327593 SRS463965 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88 / M. marinum infected GSM1195171   source name:myd88 mutant myd88 / M.marinum infected 4 dpi|sample type:myd88 mutant zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:myd88 mutant|tissue:embryo myd88 / M. marinum infected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample myd88 mutant myd88 / M.marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:myd88 mutant zebrafish embryos injected with M. marinum at 28 hpf; RNA isolated at 4 dpi|treatment:injected with M. marinum|genotype:myd88 mutant|tissue:embryo GSM1195171 GSM1195171: myd88 / M. marinum infected; Danio rerio; RNA Seq GSM1195171   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195171 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     20_ATGTCA_L001_R1_001_run2.fastq.gz 20_ATGTCA_L001_R2_001_run2.fastq.gz fastq fastq 922117740.0 9040370.0 GSM1195171 r2 0:51 1:51 A:248447288;C:217566166;G:210801103;T:244850330;N:452853 51 51     248447288 217566166 210801103 244850330 452853 SRX327593 SRS463965 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91628 0.91162 0.11324 0.1157 0.66953 0.66774 0.48076 0.47406 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37118 37118 SRR942764 SRX327592 SRS463964 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88 / uninfected GSM1195170   source name:myd88 mutant myd88 / not infected|sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo myd88 / uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample myd88 mutant myd88 / not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo GSM1195170 GSM1195170: myd88 / uninfected; Danio rerio; RNA Seq GSM1195170   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195170 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     19_AGTTCC_L005_R1_001_run1.fastq.gz 19_AGTTCC_L005_R2_001_run1.fastq.gz fastq fastq 527111520.0 5167760.0 GSM1195170 r1 0:51 1:51 A:141925057;C:123847416;G:120153488;T:140534193;N:651366 51 51     141925057 123847416 120153488 140534193 651366 SRX327592 SRS463964 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91898 0.91383 0.115 0.1173 0.67602 0.67148 0.47131 0.47342 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37119 37119 SRR942765 SRX327592 SRS463964 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88 / uninfected GSM1195170   source name:myd88 mutant myd88 / not infected|sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo myd88 / uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample myd88 mutant myd88 / not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo GSM1195170 GSM1195170: myd88 / uninfected; Danio rerio; RNA Seq GSM1195170   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195170 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     19_AGTTCC_L001_R1_001_run2.fastq.gz 19_AGTTCC_L001_R2_001_run2.fastq.gz fastq fastq 551202900.0 5403950.0 GSM1195170 r2 0:51 1:51 A:148112345;C:130297536;G:125673917;T:146845022;N:274080 51 51     148112345 130297536 125673917 146845022 274080 SRX327592 SRS463964 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.9225 0.9187 0.11483 0.11538 0.67314 0.67123 0.47354 0.47407 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37120 37120 SRR942766 SRX327592 SRS463964 SRP028229 PRJNA213244 Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis RNA seq GSE49186 Transcriptome Analysis MyD88 is an adaptor protein in Toll like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88 leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88 dependent genes. Overall design: This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents allele hu3568 van der Vaart et al. 2013 Disease models & mechanisms 6 841 854 or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection 4 dpi. The following treatment groups were used: homozygous mutants mock injected with PBS/2%PVP 4 dpi 2 wildtype siblings mock injected with PBS/2%PVP 4dpi 3 M. marinum infected homozygous mutants 4dpi 4 M. marinum infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hpf. Subsequently embryos were infected at 28 hpf by micro injecting 200 colony forming units CFU of Mycobacterium marinum Mma20 bacteria into the caudal vein or were mock injected with buffer PBS/2%PVP as a control. post injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. post the incubation period single embryos were snap frozen in liquid nitrogen and RNA was isolated for RNAseq analysis. parent bioproject:PRJNA213243 pubmed:24922577;pubmed:25247677   myd88 / uninfected GSM1195170   source name:myd88 mutant myd88 / not infected|sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo myd88 / uninfected Image analysis and base calling was done by the Illumina HCS version 1.15.1; raw data from 2 sample 20 and 3 sequencing runs samples 19 21 and 22 were combined to generate the tsv files and to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample myd88 mutant myd88 / not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 d post infection pools of 20 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:myd88 mutant zebrafish embryos mock injected with PBS at xxx hpf; RNA isolated at 4 dpi|treatment:PBS|genotype:myd88 mutant|tissue:embryo GSM1195170 GSM1195170: myd88 / uninfected; Danio rerio; RNA Seq GSM1195170   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1195170 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP028229     19_AGTTCC_L008_R1_001_run3.fastq.gz 19_AGTTCC_L008_R2_001_run3.fastq.gz fastq fastq 73803018.0 723559.0 GSM1195170 r3 0:51 1:51 A:19853585;C:17320732;G:16864909;T:19733714;N:30078 51 51     19853585 17320732 16864909 19733714 30078 SRX327592 SRS463964 SRA096049 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.92306 0.91503 0.11783 0.1182 0.67381 0.67389 0.4768 0.47693 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2013-07-24 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37909 37909 SRR1167763 SRX469456 SRS556743 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   ptpn6 morphant infected rep4 GSM1326129   tissue:zebrafish embryo injected with ptpn6 morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:ptpn6 morpholino 0.06 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi ptpn6 morphant infected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with ptpn6 morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:ptpn6 morpholino 0.06 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326129 GSM1326129: ptpn6 morphant infected rep4; Danio rerio; RNA Seq GSM1326129   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326129 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_26_ACTTGA_L005_R2_001.fastq.gz IL-12-05_26_ACTTGA_L005_R1_001.fastq.gz fastq fastq 6319438560.0 61955280.0 GSM1326129 r1 0:51 1:51 A:1718210745;C:1460888565;G:1425603166;T:1712017305;N:2718779 51 51     1718210745 1460888565 1425603166 1712017305 2718779 SRX469456 SRS556743 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.72599 0.74293 0.09205 0.09579 0.70074 0.7009 0.47487 0.47487 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37910 37910 SRR1167762 SRX469455 SRS556742 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   ptpn6 morphant uninfected rep4 GSM1326128   tissue:zebrafish embryo injected with ptpn6 morpholino not infected|morpholino injection at 1 cell stage:ptpn6 morpholino 0.06 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi ptpn6 morphant uninfected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with ptpn6 morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:ptpn6 morpholino 0.06 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326128 GSM1326128: ptpn6 morphant uninfected rep4; Danio rerio; RNA Seq GSM1326128   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326128 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_25_TTAGGC_L005_R1_001.fastq.gz IL-12-05_25_TTAGGC_L005_R2_001.fastq.gz fastq fastq 1148524692.0 11260046.0 GSM1326128 r1 0:51 1:51 A:308841968;C:270968269;G:263170238;T:304476103;N:1068114 51 51     308841968 270968269 263170238 304476103 1068114 SRX469455 SRS556742 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.90526 0.89981 0.11045 0.11095 0.6832 0.67876 0.45977 0.46165 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37911 37911 SRR1167761 SRX469454 SRS556741 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control infected rep4 GSM1326127   tissue:zebrafish embryo injected with control morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:control morpholino 0.06 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi standard control infected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.06 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326127 GSM1326127: standard control infected rep4; Danio rerio; RNA Seq GSM1326127   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326127 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_24_ATCACG_L005_R2_001.fastq.gz IL-12-05_24_ATCACG_L005_R1_001.fastq.gz fastq fastq 2366164278.0 23197689.0 GSM1326127 r1 0:51 1:51 A:639573191;C:553316835;G:538406938;T:633436794;N:1430520 51 51     639573191 553316835 538406938 633436794 1430520 SRX469454 SRS556741 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91611 0.90945 0.11564 0.11797 0.67357 0.67253 0.47654 0.47748 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37912 37912 SRR1167760 SRX469453 SRS556739 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control uninfected rep4 GSM1326126   tissue:zebrafish embryo injected with control morpholino not infected|morpholino injection at 1 cell stage:control morpholino 0.06 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi standard control uninfected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.06 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326126 GSM1326126: standard control uninfected rep4; Danio rerio; RNA Seq GSM1326126   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326126 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_23_GTGAAA_L004_R1_001.fastq.gz IL-12-05_23_GTGAAA_L004_R2_001.fastq.gz fastq fastq 1125160266.0 11030983.0 GSM1326126 r1 0:51 1:51 A:302102834;C:263590749;G:255340620;T:302565737;N:1560326 51 51     302102834 263590749 255340620 302565737 1560326 SRX469453 SRS556739 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.8956 0.89355 0.10949 0.11067 0.66168 0.66472 0.46991 0.46883 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37913 37913 SRR1167759 SRX469452 SRS556740 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control infected rep3 GSM1326125   tissue:zebrafish embryo injected with control morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi standard control infected rep3 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326125 GSM1326125: standard control infected rep3; Danio rerio; RNA Seq GSM1326125   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326125 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_16_CAGATC_L005_R2_001.fastq.gz IL-12-05_16_CAGATC_L005_R1_001.fastq.gz fastq fastq 1180291878.0 11571489.0 GSM1326125 r1 0:51 1:51 A:314857397;C:281778871;G:270994555;T:311906566;N:754489 51 51     314857397 281778871 270994555 311906566 754489 SRX469452 SRS556740 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.92573 0.92005 0.10904 0.1122 0.67318 0.66854 0.46089 0.47409 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37914 37914 SRR1167758 SRX469451 SRS556738 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control uninfected rep3 GSM1326124   tissue:zebrafish embryo injected with control morpholino not infected|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi standard control uninfected rep3 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326124 GSM1326124: standard control uninfected rep3; Danio rerio; RNA Seq GSM1326124   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326124 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_15_GCCAAT_L003_R2_001.fastq.gz IL-12-05_15_GCCAAT_L003_R1_001.fastq.gz fastq fastq 1104086046.0 10824373.0 GSM1326124 r1 0:51 1:51 A:295843963;C:261428271;G:252268202;T:293541985;N:1003625 51 51     295843963 261428271 252268202 293541985 1003625 SRX469451 SRS556738 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.92616 0.91953 0.11304 0.11617 0.67462 0.66987 0.47397 0.47242 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37915 37915 SRR1167757 SRX469450 SRS556737 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   mpeg1 morphant infected rep2 GSM1326123   tissue:zebrafish embryo injected with mpeg1 morpholino1 M. marinum infected 4 dpi|morpholino injection at 1 cell stage:mpeg1 mo1 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi mpeg1 morphant infected rep2 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with mpeg1 morpholino1 M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:mpeg1 mo1 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326123 GSM1326123: mpeg1 morphant infected rep2; Danio rerio; RNA Seq GSM1326123   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326123 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_14_ACAGTG_L003_R2_001.fastq.gz IL-12-05_14_ACAGTG_L003_R1_001.fastq.gz fastq fastq 1329118038.0 13030569.0 GSM1326123 r1 0:51 1:51 A:358564442;C:312052518;G:301710156;T:355792707;N:998215 51 51     358564442 312052518 301710156 355792707 998215 SRX469450 SRS556737 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91715 0.91117 0.1181 0.12085 0.66813 0.66371 0.47909 0.47757 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37916 37916 SRR1167756 SRX469449 SRS556736 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   mpeg1 morphant uninfected rep2 GSM1326122   tissue:zebrafish embryo injected with mpeg1 morpholino1 not infected|morpholino injection at 1 cell stage:mpeg1 mo1 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi mpeg1 morphant uninfected rep2 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with mpeg1 morpholino1 not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:mpeg1 mo1 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326122 GSM1326122: mpeg1 morphant uninfected rep2; Danio rerio; RNA Seq GSM1326122   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326122 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_13_TGACCA_L003_R2_001.fastq.gz IL-12-05_13_TGACCA_L003_R1_001.fastq.gz fastq fastq 1165066542.0 11422221.0 GSM1326122 r1 0:51 1:51 A:313409104;C:274632156;G:266493545;T:309358658;N:1173079 51 51     313409104 274632156 266493545 309358658 1173079 SRX469449 SRS556736 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.90756 0.90139 0.12374 0.12615 0.67312 0.66693 0.46638 0.46608 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37917 37917 SRR1167755 SRX469448 SRS556735 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control infected rep2 GSM1326121   tissue:zebrafish embryo injected with control morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi standard control infected rep2 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326121 GSM1326121: standard control infected rep2; Danio rerio; RNA Seq GSM1326121   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326121 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_12_CGATGT_L003_R2_001.fastq.gz IL-12-05_12_CGATGT_L003_R1_001.fastq.gz fastq fastq 1185698694.0 11624497.0 GSM1326121 r1 0:51 1:51 A:316445097;C:282007327;G:272220173;T:314001185;N:1024912 51 51     316445097 282007327 272220173 314001185 1024912 SRX469448 SRS556735 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91904 0.91382 0.12546 0.12739 0.67444 0.67026 0.47015 0.47612 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37918 37918 SRR1167754 SRX469447 SRS556734 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control uninfected rep2 GSM1326120   tissue:zebrafish embryo injected with control morpholino not infected|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi standard control uninfected rep2 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326120 GSM1326120: standard control uninfected rep2; Danio rerio; RNA Seq GSM1326120   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326120 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_11_ATTCCT_L003_R2_001.fastq.gz IL-12-05_11_ATTCCT_L003_R1_001.fastq.gz fastq fastq 1256486592.0 12318496.0 GSM1326120 r1 0:51 1:51 A:340347924;C:292509099;G:283080157;T:339504402;N:1045010 51 51     340347924 292509099 283080157 339504402 1045010 SRX469447 SRS556734 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91603 0.9114 0.12272 0.1245 0.66886 0.66764 0.46918 0.47062 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37919 37919 SRR1167753 SRX469446 SRS556733 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control infected rep1 GSM1326119   tissue:zebrafish embryo injected with control morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi standard control infected rep1 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1326119 GSM1326119: standard control infected rep1; Danio rerio; RNA Seq GSM1326119   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326119 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_2_ACTTGA_L002_R1_001.fastq.gz IL-12-05_2_ACTTGA_L002_R2_001.fastq.gz fastq fastq 1199123526.0 11756113.0 GSM1326119 r1 0:51 1:51 A:324085188;C:279539817;G:271421586;T:322631663;N:1445272 51 51     324085188 279539817 271421586 322631663 1445272 SRX469446 SRS556733 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91763 0.91305 0.11129 0.11344 0.67101 0.66458 0.47 0.47161 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
37920 37920 SRR1167752 SRX469445 SRS556732 SRP037583 PRJNA237966 Macrophage Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti Bacterial Function in Zebrafish GSE54885 Transcriptome Analysis Macrophage expressed gene 1 MPEG1 encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates MPEG1 is an integral membrane protein of macrophages but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1 two of which mpeg1 and mpeg1.2 are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down regulated during infection with both pathogens mpeg1.2 is infection inducible. Up regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll like receptor adaptor molecule MyD88 and transcription factor NF?B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection both mpeg1 and mpeg1.2 knockdown increase bacterial burdens but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti bacterial function of the Mpeg1 family and indicate that the intricate cross regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Overall design: Embryos were injected at the one cell stage with a morpholino targeting mpeg1 or with the standard control morpholino from GeneTools or with a morpholino targeting ptpn6 Kanwal et al. 2013 J. Immunol 190:1631 45 for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanisation an…   pubmed:25247677   standard control uninfected rep1 GSM1326118   tissue:zebrafish embryo injected with control morpholino not infected|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi standard control uninfected rep1 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 and 23 was performed while the raw data from 2 sequencing runs sample 25 and 3 sequencing runs samples 2 11 16 24 and 26 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Zv9 67 using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1326118 GSM1326118: standard control uninfected rep1; Danio rerio; RNA Seq GSM1326118   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1326118 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP037583     IL-12-05_1_TTAGGC_L002_R1_001.fastq.gz IL-12-05_1_TTAGGC_L002_R2_001.fastq.gz fastq fastq 1125551946.0 11034823.0 GSM1326118 r1 0:51 1:51 A:303069240;C:263328769;G:254941514;T:302623967;N:1588456 51 51     303069240 263328769 254941514 302623967 1588456 SRX469445 SRS556732 SRA139243 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91899 0.91421 0.10624 0.10789 0.6706 0.67111 0.4694 0.47206 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-02-11 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38024 38024 SRR1291421 SRX546379 SRS613409 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   M. marinum E11 5DPI [IL 13 08 Anita 39 CGATGT L001 R1 001] GSM1388565   tissue:embryo 5 dpf marinum E11 mCherry M. marinum E11 5DPI [IL 13 08 Anita 39 CGATGT L001 R1 001] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:M. marinum E11 mCherry GSM1388565 GSM1388565: M. marinum E11 5DPI [IL 13 08 Anita 39 CGATGT L001 R1 001]; Danio rerio; RNA Seq GSM1388565   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388565 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_39_CGATGT_L001_R1_001.fastq.gz fastq 821928750.0 16116250.0 GSM1388565 r1 0:51 A:218242507;C:196761189;G:188703076;T:217882057;N:339921 51       218242507 196761189 188703076 217882057 339921 SRX546379 SRS613409 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92552   0.12016   0.66823   0.47762   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38025 38025 SRR1291420 SRX546378 SRS613407 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   M. marinum E11 5DPI [IL 13 08 Anita 38 ATTCCT L002 R1 001] GSM1388564   tissue:embryo 5 dpf marinum E11 mCherry M. marinum E11 5DPI [IL 13 08 Anita 38 ATTCCT L002 R1 001] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:M. marinum E11 mCherry GSM1388564 GSM1388564: M. marinum E11 5DPI [IL 13 08 Anita 38 ATTCCT L002 R1 001]; Danio rerio; RNA Seq GSM1388564   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388564 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_38_ATTCCT_L002_R1_001.fastq.gz fastq 1415240769.0 27749819.0 GSM1388564 r1 0:51 A:378251326;C:336229238;G:324549915;T:375984079;N:226211 51       378251326 336229238 324549915 375984079 226211 SRX546378 SRS613407 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92503   0.11203   0.66488   0.4802   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38026 38026 SRR1291419 SRX546377 SRS613408 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   M. marinum E11 5DPI [IL 13 08 Anita 37 ACTGAT L007 R1 001] GSM1388563   tissue:embryo 5 dpf marinum E11 mCherry M. marinum E11 5DPI [IL 13 08 Anita 37 ACTGAT L007 R1 001] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:M. marinum E11 mCherry GSM1388563 GSM1388563: M. marinum E11 5DPI [IL 13 08 Anita 37 ACTGAT L007 R1 001]; Danio rerio; RNA Seq GSM1388563   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388563 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_37_ACTGAT_L007_R1_001.fastq.gz fastq 981130248.0 19237848.0 GSM1388563 r1 0:51 A:263661886;C:231300628;G:222568276;T:263281589;N:317869 51       263661886 231300628 222568276 263281589 317869 SRX546377 SRS613408 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92181   0.11638   0.67067   0.48179   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38027 38027 SRR1291418 SRX546376 SRS613406 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   M. marinum E11 5DPI [IL 13 08 Anita 27 ATCACG L008 R1 001] GSM1388562   tissue:embryo 5 dpf marinum E11 mCherry M. marinum E11 5DPI [IL 13 08 Anita 27 ATCACG L008 R1 001] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:M. marinum E11 mCherry GSM1388562 GSM1388562: M. marinum E11 5DPI [IL 13 08 Anita 27 ATCACG L008 R1 001]; Danio rerio; RNA Seq GSM1388562   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388562 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_27_ATCACG_L008_R1_001.fastq.gz fastq 521360454.0 10222754.0 GSM1388562 r1 0:51 A:139740762;C:122793226;G:119631427;T:138999661;N:195378 51       139740762 122793226 119631427 138999661 195378 SRX546376 SRS613406 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92116   0.12379   0.66985   0.46786   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38028 38028 SRR1291417 SRX546375 SRS613405 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   M. marinum E11 5DPI [IL 13 08 Anita 26 GTGAAA] GSM1388561   tissue:embryo 5 dpf marinum E11 mCherry M. marinum E11 5DPI [IL 13 08 Anita 26 GTGAAA] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:M. marinum E11 mCherry GSM1388561 GSM1388561: M. marinum E11 5DPI [IL 13 08 Anita 26 GTGAAA]; Danio rerio; RNA Seq GSM1388561   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388561 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_26_GTGAAA.fastq.gz fastq 825381603.0 16183953.0 GSM1388561 r1 0:51 A:222172183;C:193209766;G:188501378;T:220304536;N:1193740 51       222172183 193209766 188501378 220304536 1193740 SRX546375 SRS613405 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.91874   0.12744   0.66527   0.4704   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38029 38029 SRR1291416 SRX546374 SRS613404 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   Non injected 5 DPI [IL 13 08 Anita 7 TAGCTT] GSM1388560   tissue:embryo 5 dpf Non injected 5 DPI [IL 13 08 Anita 7 TAGCTT] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:control GSM1388560 GSM1388560: Non injected 5 DPI [IL 13 08 Anita 7 TAGCTT]; Danio rerio; RNA Seq GSM1388560   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388560 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_7_TAGCTT.fastq.gz fastq 656797941.0 12878391.0 GSM1388560 r1 0:51 A:175051093;C:155466136;G:151481640;T:174025377;N:773695 51       175051093 155466136 151481640 174025377 773695 SRX546374 SRS613404 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92338   0.10722   0.67014   0.47805   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38030 38030 SRR1291415 SRX546373 SRS613403 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   Non injected 5 DPI [IL 13 08 Anita 6 GATCAG] GSM1388559   tissue:embryo 5 dpf Non injected 5 DPI [IL 13 08 Anita 6 GATCAG] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:control GSM1388559 GSM1388559: Non injected 5 DPI [IL 13 08 Anita 6 GATCAG]; Danio rerio; RNA Seq GSM1388559   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388559 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_6_GATCAG.fastq.gz fastq 727117302.0 14257202.0 GSM1388559 r1 0:51 A:193094210;C:173124518;G:166832947;T:192824372;N:1241255 51       193094210 173124518 166832947 192824372 1241255 SRX546373 SRS613403 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92164   0.10283   0.66825   0.4689   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38031 38031 SRR1291414 SRX546372 SRS613402 SRP042084 PRJNA248106 Gene expression profiling of zebrafish embryos at 5 dpf GSE57792 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against Mycobacterium marinum. Therefore we injected M. marinum into the yolk at the 64 cell stage and took samples at 5 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by Mycobacterium marinum infection. RNA was isolated from embryos at 5 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 64 cell stage with 40 CFU of Mycobacterium marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 5 days post injection 50 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:25503064   Non injected 5 DPI [IL 13 08 Anita 5 ACTTGA] GSM1388558   tissue:embryo 5 dpf Non injected 5 DPI [IL 13 08 Anita 5 ACTTGA] GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into tsv files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: Zv9 toplevel Supplementary files format and content: txt file containing read counts per gene embryo 5 dpf Zebrafish embryos were micro injected into the yolk 64 cell stage with M. marinum E11 mCherry bacteria suspended in PVP Polyvinylpyrrolidone or Non injected as a control. At 5 days post injection 50 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. infection:control GSM1388558 GSM1388558: Non injected 5 DPI [IL 13 08 Anita 5 ACTTGA]; Danio rerio; RNA Seq GSM1388558   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM1388558 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2000   SRP042084     IL-13-08_Anita_5_ACTTGA.fastq.gz fastq 652146843.0 12787193.0 GSM1388558 r1 0:51 A:173068611;C:155658070;G:150059826;T:172627469;N:732867 51       173068611 155658070 150059826 172627469 732867 SRX546372 SRS613402 SRA165459 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92687   0.08834   0.6774   0.47425   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2014-05-19 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38038 38038 SRR1342219 SRX573023 SRS627980 SRP042975 PRJNA251728 Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco a key control factor of pro inflammatory signalling GSE58230 Transcriptome Analysis Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco macrophage receptor with collagenous structure during infection of zebrafish embryos with Mycobacterium marinum a close relative of Mycobacterium tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore gene expression analysis shows that Marco controls the initial transient pro inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth likely due to delayed phagocytosis and reduced pro inflammatory signalling observed under conditions of Marco deficiency Overall design: Embryos were injected at the one cell stage with a morpholino targeting marco or with the standard control morpholino from GeneTools for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanization and incubated for 4 days at 28°C. post the incubation period infected and uninfected morphants mutants and their controls were imaged and groups of 30 embryos were snap frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.   pubmed:25086293   marco morphant infected rep4 GSM1404384   tissue:zebrafish embryo injected with marco morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:marco morpholino 0.02 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu marco morphant infected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 was performed while the raw data from 3 sequencing runs samples 2 9 and 10 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Zv9 Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with marco morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:marco morpholino 0.02 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1404384 GSM1404384: marco morphant infected rep4; Danio rerio; RNA Seq GSM1404384   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1404384 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP042975     IL-12-05_10_ACTGAT_L003_R2_001.fastq.gz IL-12-05_10_ACTGAT_L003_R1_001.fastq.gz fastq fastq 1207891446.0 11842073.0 GSM1404384 r1 0:51 1:51 A:328473296;C:279991232;G:270373717;T:327943714;N:1109487 51 51     328473296 279991232 270373717 327943714 1109487 SRX573023 SRS627980 SRA169358 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91618 0.91223 0.12335 0.12702 0.66752 0.6649 0.4683 0.46612 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-06-04 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38039 38039 SRR1342218 SRX573022 SRS627979 SRP042975 PRJNA251728 Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco a key control factor of pro inflammatory signalling GSE58230 Transcriptome Analysis Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco macrophage receptor with collagenous structure during infection of zebrafish embryos with Mycobacterium marinum a close relative of Mycobacterium tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore gene expression analysis shows that Marco controls the initial transient pro inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth likely due to delayed phagocytosis and reduced pro inflammatory signalling observed under conditions of Marco deficiency Overall design: Embryos were injected at the one cell stage with a morpholino targeting marco or with the standard control morpholino from GeneTools for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanization and incubated for 4 days at 28°C. post the incubation period infected and uninfected morphants mutants and their controls were imaged and groups of 30 embryos were snap frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.   pubmed:25086293   marco morphant uninfected rep4 GSM1404383   tissue:zebrafish embryo injected with marco morpholino not infected|morpholino injection at 1 cell stage:marco morpholino 0.02 mM|blood island injection at 28 hpf:PBS/2%PVP marco morphant uninfected rep4 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 was performed while the raw data from 3 sequencing runs samples 2 9 and 10 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Zv9 Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with marco morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:marco morpholino 0.02 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1404383 GSM1404383: marco morphant uninfected rep4; Danio rerio; RNA Seq GSM1404383   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1404383 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP042975     IL-12-05_9_GAGTGG_L003_R1_001.fastq.gz IL-12-05_9_GAGTGG_L003_R2_001.fastq.gz fastq fastq 1248563028.0 12240814.0 GSM1404383 r1 0:51 1:51 A:337777282;C:290967808;G:281893529;T:337006324;N:918085 51 51     337777282 290967808 281893529 337006324 918085 SRX573022 SRS627979 SRA169358 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91949 0.91263 0.11016 0.11021 0.67241 0.67136 0.47526 0.47587 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-06-04 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38040 38040 SRR1342217 SRX573021 SRS627978 SRP042975 PRJNA251728 Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco a key control factor of pro inflammatory signalling GSE58230 Transcriptome Analysis Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco macrophage receptor with collagenous structure during infection of zebrafish embryos with Mycobacterium marinum a close relative of Mycobacterium tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore gene expression analysis shows that Marco controls the initial transient pro inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth likely due to delayed phagocytosis and reduced pro inflammatory signalling observed under conditions of Marco deficiency Overall design: Embryos were injected at the one cell stage with a morpholino targeting marco or with the standard control morpholino from GeneTools for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanization and incubated for 4 days at 28°C. post the incubation period infected and uninfected morphants mutants and their controls were imaged and groups of 30 embryos were snap frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.   pubmed:25086293   standard control infected rep1 GSM1404382   tissue:zebrafish embryo injected with control morpholino M. marinum infected 4 dpi|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu standard control infected rep1 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 was performed while the raw data from 3 sequencing runs samples 2 9 and 10 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Zv9 Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino M. marinum infected 4 dpi Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:M. marinum Mma20 strain 200 cfu|developmental stage rna isolation:4 dpi GSM1404382 GSM1404382: standard control infected rep1; Danio rerio; RNA Seq GSM1404382   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1404382 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP042975         1199123526.0 11756113.0 GSM1404382 r1 0:51 1:51 A:324085188;C:279539817;G:271421586;T:322631663;N:1445272 51 51     324085188 279539817 271421586 322631663 1445272 SRX573021 SRS627978 SRA169358 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91765 0.91317 0.11126 0.11356 0.67054 0.66484 0.47007 0.47218 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-06-04 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
38041 38041 SRR1342216 SRX573020 SRS627977 SRP042975 PRJNA251728 Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco a key control factor of pro inflammatory signalling GSE58230 Transcriptome Analysis Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco macrophage receptor with collagenous structure during infection of zebrafish embryos with Mycobacterium marinum a close relative of Mycobacterium tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore gene expression analysis shows that Marco controls the initial transient pro inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth likely due to delayed phagocytosis and reduced pro inflammatory signalling observed under conditions of Marco deficiency Overall design: Embryos were injected at the one cell stage with a morpholino targeting marco or with the standard control morpholino from GeneTools for comparison. Subsequently at 24 hpf the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein or mock injected with PBS/2%PVP. post injections embryos were transferred into fresh egg water containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich to prevent melanization and incubated for 4 days at 28°C. post the incubation period infected and uninfected morphants mutants and their controls were imaged and groups of 30 embryos were snap frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.   pubmed:25086293   standard control uninfected rep1 GSM1404381   tissue:zebrafish embryo injected with control morpholino not infected|morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP standard control uninfected rep1 Image analysis and base calling was done by the Illumina HCS version 1.15.1; One sequencing run for sample 1 was performed while the raw data from 3 sequencing runs samples 2 9 and 10 were combined to generate one fastq file and one tsv file each. The generated fastq files were used to calculate average insert sizes and standard deviations for paired end experiments. Sequence reads were quality trimmed using the quality trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts Danio rerio.Zv9.67.dna.toplevel / Danio rerio.Zv9.67.cdna.all.fa http://www.ensembl.org/info/data/ftp/index.html using the ref assemle short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly table module in the CLCbio Assembly Cell v4.0.6. Secondly a custom script was used that sums all reads belonging to the same gene. Non uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold change and differential expression significance values were calculated from gene level read counts using the DESeq package version 1.8.3 available in Bioconductor version 2.10. Genome build: Zv9 Supplementary files format and content: tab delimited text files include RPKM values for each Sample zebrafish embryo injected with control morpholino not infected Zebrafish embryos were manually dechorionated at 24 hpf and at 28 hpf they were micro injected into the caudal vein with 200 CFU of M. marinum Mma20 bacteria suspended in PBS/2%PVP or mock injected with PBS/2%PVP as a control. At 5 dpf 4 days post infection pools of 30 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using QIAZOL reagent. Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts containing 0.003% 1 phenyl 2 thiourea Sigma Aldrich morpholino injection at 1 cell stage:control morpholino 0.2 mM|blood island injection at 28 hpf:PBS/2%PVP|developmental stage rna isolation:4 dpi GSM1404381 GSM1404381: standard control uninfected rep1; Danio rerio; RNA Seq GSM1404381   1 Pools of embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 0 7 ml of QIAzol® Reagent Qiagen and subsequently total RNA was extracted and on column DNase digestion was performed with a miRNeasy Mini Kit Qiagen according to the manufacturer’s instructions. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. A total of 3 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 Illumina Inc. San Diego USA. In the manufacturer’s instructions two modifications were made. In the adapter ligation step 1 µl instead of 2.5 µl adapter was used. In the library size selection step the library fragments were isolated with a double Ampure XP purification with a 0.7x beads to library ratio. The resulting mRNA Seq library was sequenced using an Illumina HiSeq2000 instrument according to the manufacturer’s description with a read length of 2 x 50 nucleotides. GEO Accession:GSM1404381 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP042975         1125551946.0 11034823.0 GSM1404381 r1 0:51 1:51 A:303069240;C:263328769;G:254941514;T:302623967;N:1588456 51 51     303069240 263328769 254941514 302623967 1588456 SRX573020 SRS627977 SRA169358 GEO Animal Sciences and Health, Institute of Biology, Leiden University 2 0.91898 0.91414 0.10606 0.1084 0.66991 0.67203 0.46966 0.47223 51 51 B B biological fallback assumption illumina hiseq_era unknown poly_a trueseq bulk unknown unknown   Netherlands 2014-06-04 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40440 40440 SRR3159085 SRX1569410 SRS1282970 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 3dpi C GSM2058661   source name:Danio rerio embryo 5 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 3|tissue:embryo Nano 3dpi C GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 3|tissue:embryo GSM2058661 GSM2058661: Nano 3dpi C; Danio rerio; RNA Seq GSM2058661   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058661 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     3dpi_70nm_Polystyrene_inject_combined.fastq fastq 1137257619.0 22299169.0 GSM2058661 r1 0:51 A:307247299;C:264916697;G:256850019;T:308103905;N:139699 51       307247299 264916697 256850019 308103905 139699 SRX1569410 SRS1282970 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92017   0.1306   0.67616   0.46248   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40441 40441 SRR3159084 SRX1569409 SRS1282969 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 3dpi B GSM2058660   source name:Danio rerio embryo 5 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo Nano 3dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo GSM2058660 GSM2058660: Nano 3dpi B; Danio rerio; RNA Seq GSM2058660   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058660 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Nano_3dpi_B_ZFG-15-14_12_merged.fastq fastq 706674054.0 13856354.0 GSM2058660 r1 0:51 A:177282863;C:166514135;G:162221864;T:200591285;N:63907 51       177282863 166514135 162221864 200591285 63907 SRX1569409 SRS1282969 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92722   0.11536   0.67077   0.45483   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40442 40442 SRR3159083 SRX1569408 SRS1282971 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 3dpi A GSM2058659   source name:Danio rerio embryo 5 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo Nano 3dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo GSM2058659 GSM2058659: Nano 3dpi A; Danio rerio; RNA Seq GSM2058659   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058659 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Nano_3dpi_A_ZFG-15-14_11_merged.fastq fastq 862376544.0 16909344.0 GSM2058659 r1 0:51 A:216073645;C:202421089;G:199421641;T:244307558;N:152611 51       216073645 202421089 199421641 244307558 152611 SRX1569408 SRS1282971 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92581   0.11736   0.67006   0.45745   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40443 40443 SRR3159082 SRX1569407 SRS1282972 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Mock 3dpi B GSM2058658   source name:Danio rerio embryo 5 dpf type:Wildtype: mock injected control with PVP; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo Mock 3dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: mock injected control with PVP; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo GSM2058658 GSM2058658: Mock 3dpi B; Danio rerio; RNA Seq GSM2058658   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058658 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Mock_3dpi_B_ZFG-15-14_10_merged.fastq fastq 530494350.0 10401850.0 GSM2058658 r1 0:51 A:136358817;C:122449197;G:121498653;T:150087244;N:100439 51       136358817 122449197 121498653 150087244 100439 SRX1569407 SRS1282972 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.91253   0.12533   0.6938   0.45768   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40444 40444 SRR3159081 SRX1569406 SRS1282973 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Mock 3dpi A GSM2058657   source name:Danio rerio embryo 5 dpf type:Wildtype: mock injected control with PVP; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo Mock 3dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: mock injected control with PVP; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo GSM2058657 GSM2058657: Mock 3dpi A; Danio rerio; RNA Seq GSM2058657   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058657 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Mock_3dpi_A_ZFG-15-14_9_merged.fastq fastq 789858828.0 15487428.0 GSM2058657 r1 0:51 A:198354184;C:188071343;G:183001069;T:220292329;N:139903 51       198354184 188071343 183001069 220292329 139903 SRX1569406 SRS1282973 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.91801   0.09627   0.71691   0.45797   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40445 40445 SRR3159080 SRX1569405 SRS1282975 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 3dpi C GSM2058656   source name:Danio rerio embryo 5 dpf type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 3|tissue:embryo Control 3dpi C GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 3|tissue:embryo GSM2058656 GSM2058656: Control 3dpi C; Danio rerio; RNA Seq GSM2058656   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058656 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     3dpi_Non-injected_control_combined.fastq fastq 1244713446.0 24406146.0 GSM2058656 r1 0:51 A:338230454;C:287626139;G:278652890;T:340051827;N:152136 51       338230454 287626139 278652890 340051827 152136 SRX1569405 SRS1282975 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92117   0.13951   0.67474   0.46536   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40446 40446 SRR3159079 SRX1569404 SRS1282974 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 3dpi B GSM2058655   source name:Danio rerio embryo 5 dpf type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo Control 3dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 2|tissue:embryo GSM2058655 GSM2058655: Control 3dpi B; Danio rerio; RNA Seq GSM2058655   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058655 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Control_3dpi_B_ZFG-15-14_8_merged.fastq fastq 546552261.0 10716711.0 GSM2058655 r1 0:51 A:136953155;C:128224594;G:127888976;T:153436215;N:49321 51       136953155 128224594 127888976 153436215 49321 SRX1569404 SRS1282974 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92597   0.11002   0.67312   0.45376   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40447 40447 SRR3159078 SRX1569403 SRS1282976 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 3dpi A GSM2058654   source name:Danio rerio embryo 5 dpf type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo Control 3dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 5 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 5 dpf = 3dpi timepoint. Biological replica 1|tissue:embryo GSM2058654 GSM2058654: Control 3dpi A; Danio rerio; RNA Seq GSM2058654   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058654 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Control_3dpi_A_ZFG-15-14_7_merged.fastq fastq 704386755.0 13811505.0 GSM2058654 r1 0:51 A:181197573;C:163283607;G:160840189;T:198938688;N:126698 51       181197573 163283607 160840189 198938688 126698 SRX1569403 SRS1282976 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92252   0.12074   0.68594   0.46646   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40448 40448 SRR3159077 SRX1569402 SRS1282979 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 1dpi C GSM2058653   source name:Danio rerio embryo 3 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 3|tissue:embryo Nano 1dpi C GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 3|tissue:embryo GSM2058653 GSM2058653: Nano 1dpi C; Danio rerio; RNA Seq GSM2058653   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058653 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     1dpi_70nm_Polystyrene_inject_combined.fastq fastq 1019336133.0 19986983.0 GSM2058653 r1 0:51 A:276086303;C:236965090;G:228985025;T:277173913;N:125802 51       276086303 236965090 228985025 277173913 125802 SRX1569402 SRS1282979 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92053   0.13768   0.68444   0.4662   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40449 40449 SRR3159076 SRX1569401 SRS1282978 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 1dpi B GSM2058652   source name:Danio rerio embryo 3 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo Nano 1dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo GSM2058652 GSM2058652: Nano 1dpi B; Danio rerio; RNA Seq GSM2058652   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058652 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Nano_1dpi_B_ZFG-15-14_6_merged.fastq fastq 873629235.0 17129985.0 GSM2058652 r1 0:51 A:224250216;C:202455257;G:198589503;T:248183437;N:150822 51       224250216 202455257 198589503 248183437 150822 SRX1569401 SRS1282978 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92462   0.13401   0.67499   0.45958   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40450 40450 SRR3159075 SRX1569400 SRS1282977 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Nano 1dpi A GSM2058651   source name:Danio rerio embryo 3 dpf type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo Nano 1dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype; injected with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles into the yolk at 2 dpf; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo GSM2058651 GSM2058651: Nano 1dpi A; Danio rerio; RNA Seq GSM2058651   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058651 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Nano_1dpi_A_ZFG-15-14_5_merged.fastq fastq 812876964.0 15938764.0 GSM2058651 r1 0:51 A:208377625;C:187809751;G:185819753;T:230799242;N:70593 51       208377625 187809751 185819753 230799242 70593 SRX1569400 SRS1282977 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92654   0.12297   0.66582   0.4673   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40451 40451 SRR3159074 SRX1569399 SRS1282980 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Mock 1dpi B GSM2058650   source name:Danio rerio embryo 3 dpf type:Wildtype: mock injected control with PVP; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo Mock 1dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: mock injected control with PVP; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo GSM2058650 GSM2058650: Mock 1dpi B; Danio rerio; RNA Seq GSM2058650   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058650 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Mock_1dpi_B_ZFG-15-14_4_merged.fastq fastq 756707349.0 14837399.0 GSM2058650 r1 0:51 A:190997880;C:177597704;G:174435131;T:213606760;N:69874 51       190997880 177597704 174435131 213606760 69874 SRX1569399 SRS1282980 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92772   0.11384   0.66882   0.46142   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40452 40452 SRR3159073 SRX1569398 SRS1282981 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Mock 1dpi A GSM2058649   source name:Danio rerio embryo 3 dpf type:Wildtype: mock injected control with PVP; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo Mock 1dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: mock injected control with PVP; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo GSM2058649 GSM2058649: Mock 1dpi A; Danio rerio; RNA Seq GSM2058649   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058649 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Mock_1dpi_A_ZFG-15-14_3_merged.fastq fastq 814358616.0 15967816.0 GSM2058649 r1 0:51 A:206515446;C:189866386;G:185439892;T:232375021;N:161871 51       206515446 189866386 185439892 232375021 161871 SRX1569398 SRS1282981 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92746   0.12022   0.66446   0.46004   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40453 40453 SRR3159072 SRX1569397 SRS1282982 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 1dpi C GSM2058648   source name:Danio rerio embryo 3 dpf type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo Control 1dpi C GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo GSM2058648 GSM2058648: Control 1dpi C; Danio rerio; RNA Seq GSM2058648   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058648 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     1dpi_Non-injected_control_combined.fastq fastq 1182868041.0 23193491.0 GSM2058648 r1 0:51 A:322106653;C:272994984;G:265110490;T:322515182;N:140732 51       322106653 272994984 265110490 322515182 140732 SRX1569397 SRS1282982 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92161   0.13798   0.69189   0.47221   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40454 40454 SRR3159071 SRX1569396 SRS1282983 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 1dpi B GSM2058647   source name:Danio rerio embryo 3 dpf type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo Control 1dpi B GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 2|tissue:embryo GSM2058647 GSM2058647: Control 1dpi B; Danio rerio; RNA Seq GSM2058647   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058647 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Control_1dpi_B_ZFG-15-14_2_merged.fastq fastq 553430733.0 10851583.0 GSM2058647 r1 0:51 A:141662557;C:128215443;G:125747312;T:157707455;N:97966 51       141662557 128215443 125747312 157707455 97966 SRX1569396 SRS1282983 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92467   0.13257   0.67813   0.45716   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
40455 40455 SRR3159070 SRX1569395 SRS1282984 SRP069839 PRJNA311350 Marker gene/pathway discovery for polystyrene particle toxicity in zebrafish larvae GSE77755 Transcriptome Analysis We use the zebrafish embryo model to study the innate immune response against polystyrene particles. Therefore we injected 700nm polystyrene into the yolk at 2 dpf and took samples at xxx and 3 days post injection. Overall design: This deep sequence study was designed to determine the gene expression profile by polystyrene particle toxicity. RNA was isolated from embryos at 1 and 3 days post injection. Wildtypes zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 mock injected with pvp n=2 or Non injected as a control n=3. post injections embryos were transferred into fresh egg water and incubated at 28°C. At 1 and 3 days post injection 10 embryos per group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent.   pubmed:28704660   Control 1dpi A GSM2058646   source name:Danio rerio embryo 3 dpf type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo Control 1dpi A GeneTiles was used for quantification and visualization of the RNAseq data Bowtie2 is used to align the reads in the fastq file to the genome obtained from Ensemble. Bowtie2 generates SAM files that contain the reads together with the location on the genome. Samtools is used to convert and compress the SAM files into a binary BAM file. Samtools is furthermore used to sort the reads in the BAM files based on the aligned read location in the genome resulting in a sorted BAM file. The BAM files is indexed to be able to quickly find the aligned reads based on a location in the genome i.e. to be able to quickly search the BAM file. The index is a saved as a BAI file. Using the available annotation from Ensemble we can search the BAM file for reads within a gene using exon starting positions and lengths. This is done with a python script. The output of this script is a tab separated file tsv containing the read counts per gene. We used DESeq an R script to perform statistical analysis. The DESeq script is used to normalise the reads per gene based on divided by the total number of reads obtained per sample. Then variance and average of the measurement compared to the control can be expressed as a P value by calculating the dispersion per gene using DESeq. The size factors as well as the P values are stored in ‘tsv’ files. Using a script all tsv files are combined into xlsx files e.g. per experiment chromosome per filtered results of most significant reads or highest ratio between measurement and control. Genome build: GRCz10 Supplementary files format and content: counts Danio rerio embryo 3 dpf Zebrafish embryos were micro injected into the yolk 2dpf with 1nl of 5mg/ml of 700nm red fluorescent polystyrene particles suspended in PVP Polyvinylpyrrolidone n=3 or mock injected n=2 containting PVP only or Non injected as a control n=3. At 1 and 3 days post injection 3 dpf and 5 dpf 10 embryos per treatment group were snap frozen in liquid nitrogen and total RNA was isolated using TRIZOL reagent. Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols http://ZFIN.org. Embryos were grown at 28 5°C in egg water 60µg/ml Instant Ocean sea salts. sample type:Wildtype: non injected control; RNA isolated at 3 dpf = 1dpi timepoint. Biological replica 1|tissue:embryo GSM2058646 GSM2058646: Control 1dpi A; Danio rerio; RNA Seq GSM2058646   1 Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at 80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent Invitrogen and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI Roche Applied Science to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit Qiagen according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab on chip analysis using the 2100 Bioanalyzer Agilent Technologies. RNA libraries were prepared for sequencing using standard Illumina protocols GEO Accession:GSM2058646 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP069839     Control_1dpi_A_ZFG-15-14_1_merged.fastq fastq 737944092.0 14469492.0 GSM2058646 r1 0:51 A:186707828;C:172934142;G:169575851;T:208595646;N:130625 51       186707828 172934142 169575851 208595646 130625 SRX1569395 SRS1282984 SRA352578 GEO Molecular Cell Biology, Institute Biology Leiden, Leiden University 1 0.92761   0.12419   0.67294   0.46178   51   B   usable mapping rate illumina hiseq_era unknown cdna_unspecified unknown bulk unknown unknown   Netherlands 2016-02-09 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41092 41092 SRR3679556 SRX1852543 SRS1509724 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Water rep 3 GSM2203921   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Water rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203921 GSM2203921: 96 hpf Nfe2 KO Water rep 3; Danio rerio; RNA Seq GSM2203921   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203921 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_water_3_fwd.fastq.gz 96_Nfe2KO_water_3_rev.fastq.gz fastq fastq 2666869800.0 26668698.0 GSM2203921 r1 0:50 1:50 A:737776704;C:592046608;G:597619233;T:737576762;N:1850493 50 50     737776704 592046608 597619233 737576762 1850493 SRX1852543 SRS1509724 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93641 0.94161 0.13311 0.13058 0.68168 0.6816 0.48066 0.48079 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41093 41093 SRR3679555 SRX1852542 SRS1509723 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Water rep 2 GSM2203920   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Water rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203920 GSM2203920: 96 hpf Nfe2 KO Water rep 2; Danio rerio; RNA Seq GSM2203920   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203920 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_water_2_fwd.fastq.gz 96_Nfe2KO_water_2_rev.fastq.gz fastq fastq 2750298700.0 27502987.0 GSM2203920 r1 0:50 1:50 A:754379615;C:617146433;G:621887266;T:754993275;N:1892111 50 50     754379615 617146433 621887266 754993275 1892111 SRX1852542 SRS1509723 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93906 0.94282 0.12854 0.1264 0.68274 0.68237 0.47478 0.47562 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41094 41094 SRR3679554 SRX1852541 SRS1509722 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Water rep 1 GSM2203919   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Water rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203919 GSM2203919: 96 hpf Nfe2 KO Water rep 1; Danio rerio; RNA Seq GSM2203919   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203919 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_water_1_rev.fastq.gz 96_Nfe2KO_water_1_fwd.fastq.gz fastq fastq 2701769300.0 27017693.0 GSM2203919 r1 0:50 1:50 A:747756992;C:598626280;G:605483402;T:747647078;N:2255548 50 50     747756992 598626280 605483402 747647078 2255548 SRX1852541 SRS1509722 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93541 0.94023 0.13806 0.13564 0.68532 0.68576 0.47524 0.48386 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41095 41095 SRR3679553 SRX1852540 SRS1509721 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO tBOOH rep 3 GSM2203918   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO tBOOH rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203918 GSM2203918: 96 hpf Nfe2 KO tBOOH rep 3; Danio rerio; RNA Seq GSM2203918   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203918 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_tBOOH_3_fwd.fastq.gz 96_Nfe2KO_tBOOH_3_rev.fastq.gz fastq fastq 2511246500.0 25112465.0 GSM2203918 r1 0:50 1:50 A:692475935;C:560147617;G:564724229;T:692539061;N:1359658 50 50     692475935 560147617 564724229 692539061 1359658 SRX1852540 SRS1509721 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93618 0.94207 0.13079 0.12916 0.67986 0.67836 0.48159 0.48511 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41096 41096 SRR3679552 SRX1852539 SRS1509720 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO tBOOH rep 2 GSM2203917   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO tBOOH rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203917 GSM2203917: 96 hpf Nfe2 KO tBOOH rep 2; Danio rerio; RNA Seq GSM2203917   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203917 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_tBOOH_2_fwd.fastq.gz 96_Nfe2KO_tBOOH_2_rev.fastq.gz fastq fastq 2625379700.0 26253797.0 GSM2203917 r1 0:50 1:50 A:722017878;C:587029635;G:592273255;T:722287279;N:1771653 50 50     722017878 587029635 592273255 722287279 1771653 SRX1852539 SRS1509720 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93619 0.94156 0.1313 0.1293 0.68402 0.6831 0.48305 0.47772 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41097 41097 SRR3679551 SRX1852538 SRS1509719 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO tBOOH rep 1 GSM2203916   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO tBOOH rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203916 GSM2203916: 96 hpf Nfe2 KO tBOOH rep 1; Danio rerio; RNA Seq GSM2203916   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203916 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_tBOOH_1_fwd.fastq.gz 96_Nfe2KO_tBOOH_1_rev.fastq.gz fastq fastq 2527037300.0 25270373.0 GSM2203916 r1 0:50 1:50 A:690534490;C:569181363;G:576690235;T:689176873;N:1454339 50 50     690534490 569181363 576690235 689176873 1454339 SRX1852538 SRS1509719 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93903 0.9446 0.11507 0.11359 0.68292 0.68294 0.47665 0.48064 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41098 41098 SRR3679550 SRX1852537 SRS1509718 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Diquat rep 3 GSM2203915   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Diquat rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203915 GSM2203915: 96 hpf Nfe2 KO Diquat rep 3; Danio rerio; RNA Seq GSM2203915   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203915 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_diquat_3_fwd.fastq.gz 96_Nfe2KO_diquat_3_rev.fastq.gz fastq fastq 3102978000.0 31029780.0 GSM2203915 r1 0:50 1:50 A:866897962;C:679421656;G:689513497;T:865275974;N:1868911 50 50     866897962 679421656 689513497 865275974 1868911 SRX1852537 SRS1509718 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.9342 0.9392 0.14922 0.14651 0.6829 0.68481 0.48006 0.48767 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41099 41099 SRR3679549 SRX1852536 SRS1509717 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Diquat rep 2 GSM2203914   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Diquat rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203914 GSM2203914: 96 hpf Nfe2 KO Diquat rep 2; Danio rerio; RNA Seq GSM2203914   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203914 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_diquat_2_fwd.fastq.gz 96_Nfe2KO_diquat_2_rev.fastq.gz fastq fastq 2664048700.0 26640487.0 GSM2203914 r1 0:50 1:50 A:714190091;C:613161783;G:618783049;T:715930181;N:1983596 50 50     714190091 613161783 618783049 715930181 1983596 SRX1852536 SRS1509717 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93745 0.94256 0.11979 0.11998 0.67543 0.67517 0.45822 0.46309 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41100 41100 SRR3679548 SRX1852535 SRS1509716 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf Nfe2 KO Diquat rep 1 GSM2203913   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 96 hpf Nfe2 KO Diquat rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203913 GSM2203913: 96 hpf Nfe2 KO Diquat rep 1; Danio rerio; RNA Seq GSM2203913   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203913 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_Nfe2KO_diquat_1_fwd.fastq.gz 96_Nfe2KO_diquat_1_rev.fastq.gz fastq fastq 2762570700.0 27625707.0 GSM2203913 r1 0:50 1:50 A:753767045;C:623240109;G:628566265;T:755548665;N:1448616 50 50     753767045 623240109 628566265 755548665 1448616 SRX1852535 SRS1509716 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93846 0.94466 0.12611 0.12619 0.67598 0.67574 0.48078 0.48527 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41101 41101 SRR3679547 SRX1852534 SRS1509715 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Water rep 3 GSM2203912   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Water rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203912 GSM2203912: 96 hpf AB Water rep 3; Danio rerio; RNA Seq GSM2203912   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203912 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_water_3_fwd.fastq.gz 96_AB_water_3_rev.fastq.gz fastq fastq 2747178200.0 27471782.0 GSM2203912 r1 0:50 1:50 A:740389297;C:628048152;G:631988094;T:745197552;N:1555105 50 50     740389297 628048152 631988094 745197552 1555105 SRX1852534 SRS1509715 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94361 0.94723 0.10954 0.10892 0.65896 0.66119 0.47545 0.47595 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41102 41102 SRR3679546 SRX1852533 SRS1509714 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Water rep 2 GSM2203911   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Water rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203911 GSM2203911: 96 hpf AB Water rep 2; Danio rerio; RNA Seq GSM2203911   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203911 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_water_2_fwd.fastq.gz 96_AB_water_2_rev.fastq.gz fastq fastq 3031148700.0 30311487.0 GSM2203911 r1 0:50 1:50 A:811094501;C:697767578;G:704192577;T:816378606;N:1715438 50 50     811094501 697767578 704192577 816378606 1715438 SRX1852533 SRS1509714 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94513 0.94949 0.10658 0.10671 0.6566 0.65853 0.47216 0.47686 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41103 41103 SRR3679545 SRX1852532 SRS1509713 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Water rep 1 GSM2203910   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Water rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203910 GSM2203910: 96 hpf AB Water rep 1; Danio rerio; RNA Seq GSM2203910   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203910 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_water_1_fwd.fastq.gz 96_AB_water_1_rev.fastq.gz fastq fastq 2618962600.0 26189626.0 GSM2203910 r1 0:50 1:50 A:700789667;C:601654497;G:608275063;T:706011911;N:2231462 50 50     700789667 601654497 608275063 706011911 2231462 SRX1852532 SRS1509713 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94406 0.94993 0.10563 0.10664 0.65687 0.66107 0.47756 0.48597 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41104 41104 SRR3679544 SRX1852531 SRS1509712 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB tBOOH rep 3 GSM2203909   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB tBOOH rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203909 GSM2203909: 96 hpf AB tBOOH rep 3; Danio rerio; RNA Seq GSM2203909   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203909 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_tBOOH_3_fwd.fastq.gz 96_AB_tBOOH_3_rev.fastq.gz fastq fastq 2788758200.0 27887582.0 GSM2203909 r1 0:50 1:50 A:750029097;C:638253410;G:643990063;T:754994501;N:1491129 50 50     750029097 638253410 643990063 754994501 1491129 SRX1852531 SRS1509712 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94339 0.94767 0.10274 0.10224 0.66085 0.66166 0.47568 0.47891 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41105 41105 SRR3679543 SRX1852530 SRS1509711 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB tBOOH rep 2 GSM2203908   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB tBOOH rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203908 GSM2203908: 96 hpf AB tBOOH rep 2; Danio rerio; RNA Seq GSM2203908   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203908 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_tBOOH_2_fwd.fastq.gz 96_AB_tBOOH_2_rev.fastq.gz fastq fastq 2695465800.0 26954658.0 GSM2203908 r1 0:50 1:50 A:724342749;C:617773020;G:622527009;T:728708114;N:2114908 50 50     724342749 617773020 622527009 728708114 2114908 SRX1852530 SRS1509711 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94393 0.94808 0.1087 0.10901 0.66273 0.66289 0.48071 0.4705 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41106 41106 SRR3679542 SRX1852529 SRS1509710 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB tBOOH rep 1 GSM2203907   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB tBOOH rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203907 GSM2203907: 96 hpf AB tBOOH rep 1; Danio rerio; RNA Seq GSM2203907   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203907 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_tBOOH_1_fwd.fastq.gz 96_AB_tBOOH_1_rev.fastq.gz fastq fastq 2627127900.0 26271279.0 GSM2203907 r1 0:50 1:50 A:707681705;C:602003924;G:604784299;T:711247492;N:1410480 50 50     707681705 602003924 604784299 711247492 1410480 SRX1852529 SRS1509710 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94357 0.94735 0.10844 0.10882 0.66202 0.66336 0.47881 0.48294 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41107 41107 SRR3679541 SRX1852528 SRS1509709 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Diquat rep 3 GSM2203906   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Diquat rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203906 GSM2203906: 96 hpf AB Diquat rep 3; Danio rerio; RNA Seq GSM2203906   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203906 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_diquat_3_fwd.fastq.gz 96_AB_diquat_3_rev.fastq.gz fastq fastq 2667919100.0 26679191.0 GSM2203906 r1 0:50 1:50 A:719038633;C:610385113;G:614480088;T:721964696;N:2050570 50 50     719038633 610385113 614480088 721964696 2050570 SRX1852528 SRS1509709 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94342 0.94796 0.10582 0.10548 0.65865 0.65888 0.47541 0.48238 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41108 41108 SRR3679540 SRX1852527 SRS1509708 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Diquat rep 2 GSM2203905   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Diquat rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203905 GSM2203905: 96 hpf AB Diquat rep 2; Danio rerio; RNA Seq GSM2203905   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203905 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_diquat_2_fwd.fastq.gz 96_AB_diquat_2_rev.fastq.gz fastq fastq 2922210900.0 29222109.0 GSM2203905 r1 0:50 1:50 A:787614443;C:668655271;G:673472999;T:790874783;N:1593404 50 50     787614443 668655271 673472999 790874783 1593404 SRX1852527 SRS1509708 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94396 0.94807 0.1044 0.10356 0.65924 0.66066 0.48276 0.48013 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41109 41109 SRR3679539 SRX1852526 SRS1509707 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   96 hpf AB Diquat rep 1 GSM2203904   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf.|developmental stage:embryo|genotype:wildtype 96 hpf AB Diquat rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 20 μM Diquat. RNA sampled at 96 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203904 GSM2203904: 96 hpf AB Diquat rep 1; Danio rerio; RNA Seq GSM2203904   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203904 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     96_AB_diquat_1_fwd.fastq.gz 96_AB_diquat_1_rev.fastq.gz fastq fastq 2645304700.0 26453047.0 GSM2203904 r1 0:50 1:50 A:715909774;C:602096071;G:606636895;T:718880849;N:1781111 50 50     715909774 602096071 606636895 718880849 1781111 SRX1852526 SRS1509707 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94334 0.94769 0.10633 0.10445 0.65884 0.65985 0.47242 0.46865 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41110 41110 SRR3679538 SRX1852525 SRS1509706 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Water rep 3 GSM2203903   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Water rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203903 GSM2203903: 48 hpf Nfe2 KO Water rep 3; Danio rerio; RNA Seq GSM2203903   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203903 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_water_3_fwd.fastq.gz 48_Nfe2KO_water_3_rev.fastq.gz fastq fastq 2967132500.0 29671325.0 GSM2203903 r1 0:50 1:50 A:820170416;C:659618166;G:665536994;T:820131225;N:1675699 50 50     820170416 659618166 665536994 820131225 1675699 SRX1852525 SRS1509706 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93836 0.94354 0.12445 0.1228 0.69193 0.69313 0.47313 0.48558 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41111 41111 SRR3679537 SRX1852524 SRS1509705 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Water rep 2 GSM2203902   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Water rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203902 GSM2203902: 48 hpf Nfe2 KO Water rep 2; Danio rerio; RNA Seq GSM2203902   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203902 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_water_2_fwd.fastq.gz 48_Nfe2KO_water_2_rev.fastq.gz fastq fastq 2813832600.0 28138326.0 GSM2203902 r1 0:50 1:50 A:755860223;C:647558987;G:651096279;T:757791193;N:1525918 50 50     755860223 647558987 651096279 757791193 1525918 SRX1852524 SRS1509705 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94566 0.95116 0.09352 0.09217 0.68617 0.68564 0.47485 0.46018 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41112 41112 SRR3679536 SRX1852523 SRS1509704 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Water rep 1 GSM2203901   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Water rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203901 GSM2203901: 48 hpf Nfe2 KO Water rep 1; Danio rerio; RNA Seq GSM2203901   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203901 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_water_1_fwd.fastq.gz 48_Nfe2KO_water_1_rev.fastq.gz fastq fastq 2393386900.0 23933869.0 GSM2203901 r1 0:50 1:50 A:678752467;C:515278633;G:520182923;T:676763898;N:2408979 50 50     678752467 515278633 520182923 676763898 2408979 SRX1852523 SRS1509704 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.9321 0.93787 0.14645 0.14274 0.69852 0.69929 0.48218 0.48637 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41113 41113 SRR3679535 SRX1852522 SRS1509703 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO tBOOH rep 3 GSM2203900   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO tBOOH rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203900 GSM2203900: 48 hpf Nfe2 KO tBOOH rep 3; Danio rerio; RNA Seq GSM2203900   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203900 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_tBOOH_3_fwd.fastq.gz 48_Nfe2KO_tBOOH_3_rev.fastq.gz fastq fastq 2701357200.0 27013572.0 GSM2203900 r1 0:50 1:50 A:719851778;C:625764563;G:630626762;T:722516820;N:2597277 50 50     719851778 625764563 630626762 722516820 2597277 SRX1852522 SRS1509703 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94617 0.95317 0.09255 0.09281 0.68252 0.68592 0.46207 0.46326 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41114 41114 SRR3679534 SRX1852521 SRS1509702 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO tBOOH rep 2 GSM2203899   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO tBOOH rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203899 GSM2203899: 48 hpf Nfe2 KO tBOOH rep 2; Danio rerio; RNA Seq GSM2203899   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203899 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_tBOOH_2_fwd.fastq.gz 48_Nfe2KO_tBOOH_2_rev.fastq.gz fastq fastq 2633273000.0 26332730.0 GSM2203899 r1 0:50 1:50 A:700442742;C:611861057;G:616096300;T:702937001;N:1935900 50 50     700442742 611861057 616096300 702937001 1935900 SRX1852521 SRS1509702 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94812 0.95349 0.08888 0.08847 0.68191 0.68329 0.46644 0.45981 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41115 41115 SRR3679533 SRX1852520 SRS1509701 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO tBOOH rep 1 GSM2203898   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO tBOOH rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203898 GSM2203898: 48 hpf Nfe2 KO tBOOH rep 1; Danio rerio; RNA Seq GSM2203898   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203898 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_tBOOH_1_fwd.fastq.gz 48_Nfe2KO_tBOOH_1_rev.fastq.gz fastq fastq 2689778900.0 26897789.0 GSM2203898 r1 0:50 1:50 A:750116133;C:590806972;G:598544623;T:748820937;N:1490235 50 50     750116133 590806972 598544623 748820937 1490235 SRX1852520 SRS1509701 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93731 0.9437 0.13271 0.12966 0.70216 0.70067 0.47722 0.48919 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41116 41116 SRR3679532 SRX1852519 SRS1509700 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Diquat rep 3 GSM2203897   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Diquat rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203897 GSM2203897: 48 hpf Nfe2 KO Diquat rep 3; Danio rerio; RNA Seq GSM2203897   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203897 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_diquat_3_fwd.fastq.gz 48_Nfe2KO_diquat_3_rev.fastq.gz fastq fastq 2530493600.0 25304936.0 GSM2203897 r1 0:50 1:50 A:679215661;C:582486154;G:586880434;T:680495388;N:1415963 50 50     679215661 582486154 586880434 680495388 1415963 SRX1852519 SRS1509700 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94507 0.95025 0.10449 0.10388 0.68633 0.68667 0.46569 0.46705 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41117 41117 SRR3679531 SRX1852518 SRS1509699 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Diquat rep 2 GSM2203896   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Diquat rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203896 GSM2203896: 48 hpf Nfe2 KO Diquat rep 2; Danio rerio; RNA Seq GSM2203896   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203896 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_diquat_2_fwd.fastq.gz 48_Nfe2KO_diquat_2_rev.fastq.gz fastq fastq 2587226600.0 25872266.0 GSM2203896 r1 0:50 1:50 A:716568333;C:573809787;G:579818952;T:715639247;N:1390281 50 50     716568333 573809787 579818952 715639247 1390281 SRX1852518 SRS1509699 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.93808 0.94394 0.1285 0.1261 0.69556 0.69475 0.47281 0.48235 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41118 41118 SRR3679530 SRX1852517 SRS1509698 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf Nfe2 KO Diquat rep 1 GSM2203895   tissue:30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:Nfe2 knock out 48 hpf Nfe2 KO Diquat rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish Nfe2 knock out embryos AB background exposed to 20 μM Diquat. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:Nfe2 knock out GSM2203895 GSM2203895: 48 hpf Nfe2 KO Diquat rep 1; Danio rerio; RNA Seq GSM2203895   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203895 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_Nfe2KO_diquat_1_fwd.fastq.gz 48_Nfe2KO_diquat_1_rev.fastq.gz fastq fastq 2777659000.0 27776590.0 GSM2203895 r1 0:50 1:50 A:747538012;C:636325899;G:642869083;T:749460590;N:1465416 50 50     747538012 636325899 642869083 749460590 1465416 SRX1852517 SRS1509698 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94578 0.95075 0.10123 0.10065 0.69045 0.69029 0.45626 0.46782 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41119 41119 SRR3679529 SRX1852516 SRS1509697 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf AB Water rep 3 GSM2203894   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:wildtype 48 hpf AB Water rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203894 GSM2203894: 48 hpf AB Water rep 3; Danio rerio; RNA Seq GSM2203894   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203894 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_AB_water_3_fwd.fastq.gz 48_AB_water_3_rev.fastq.gz fastq fastq 2631850200.0 26318502.0 GSM2203894 r1 0:50 1:50 A:711636354;C:599485378;G:605519526;T:713093597;N:2115345 50 50     711636354 599485378 605519526 713093597 2115345 SRX1852516 SRS1509697 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94701 0.95156 0.10131 0.10016 0.6911 0.69073 0.45923 0.46698 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41120 41120 SRR3679528 SRX1852515 SRS1509696 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf AB Water rep 2 GSM2203893   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:wildtype 48 hpf AB Water rep 2 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203893 GSM2203893: 48 hpf AB Water rep 2; Danio rerio; RNA Seq GSM2203893   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203893 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_AB_water_2_fwd.fastq.gz 48_AB_water_2_rev.fastq.gz fastq fastq 2326803100.0 23268031.0 GSM2203893 r1 0:50 1:50 A:620107102;C:538915065;G:544301586;T:621003798;N:2475549 50 50     620107102 538915065 544301586 621003798 2475549 SRX1852515 SRS1509696 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94898 0.95295 0.09515 0.09491 0.69595 0.69601 0.45156 0.45111 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41121 41121 SRR3679527 SRX1852514 SRS1509695 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf AB Water rep 1 GSM2203892   tissue:30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:wildtype 48 hpf AB Water rep 1 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background water controls. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203892 GSM2203892: 48 hpf AB Water rep 1; Danio rerio; RNA Seq GSM2203892   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203892 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_AB_water_1_fwd.fastq.gz 48_AB_water_1_rev.fastq.gz fastq fastq 2621649300.0 26216493.0 GSM2203892 r1 0:50 1:50 A:705011318;C:600473198;G:606512433;T:707528237;N:2124114 50 50     705011318 600473198 606512433 707528237 2124114 SRX1852514 SRS1509695 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94746 0.95122 0.09964 0.09836 0.69132 0.6911 0.46414 0.46643 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures
41122 41122 SRR3679526 SRX1852513 SRS1509694 SRP076704 PRJNA326068 The transcription factor Nuclear factor erythoid 2 Nfe2 is a regulator of the oxidative stress response during Danio rerio development GSE83466 Transcriptome Analysis Development is a complex and well defined process characterized by rapid cell proliferation and apoptosis. At this stage in life a developmentally young organism is more sensitive to toxicants and other stressors when compared to an adult. In response to pro oxidant exposure members of the Cap’n’Collar CNC basic leucine zipper b ZIP transcription factor family including the Nfe2 related factors Nrfs activate the expression of genes that contribute to reduced toxicity. Here we studied the role of the Nrf protein Nfe2 in the developmental response to pro oxidant exposure in the zebrafish. Following acute waterborne exposures to diquat or tert buytlhydroperoxide tBOOH at three developmental stages wildtype WT and nfe2 knockout KO embryos and larvae were morphologically scored and their transcriptomes sequenced. Overall design: Wildtype animals were on the AB background and an additional germline nfe2 knockout strain were created by disruption of the nfe2 reading frame. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period. Total RNA was isolated from pooled animals and 50 bp paired end libraries were sequenced using the Illumina HiSeq 2000 platform with approximately 25 million reads per sample. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 and raw counts data normalized using DESeq2. Gene annotation was from Ensemble for GRCz10.   pubmed:27716579   48 hpf AB tBOOH rep 3 GSM2203891   tissue:30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf.|developmental stage:embryo|genotype:wildtype 48 hpf AB tBOOH rep 3 Raw RNA Sequencing RNA Seq reads were obtained from HudsonAlpha as 50bp paired end reads in FASTQ format with approximately 25 million reads per sample. Reads were analyzed for various quality control parameters including: per base sequence quality per sequence GC content sequence length distribution and sequence duplication levels and Kmer content using the FastQC tool Version 0.11.4. Reads were then aligned to the Ensembl GRCz10 zebrafish reference genome using Tophat2 Version 2.0.14 which functions based on Bowtie2 alignment software Version 2.2.5 using a mean inner distance of 300 bp a maximum intron length of 380 000bp and all other parameters set to default values. Read counts for each gene were then obtained for the aligned reads using the Htseq counts tool Version 0.6.1. The Tophat accepted hits BAM file was analyzed against the Ensembl GTF zebrafish gene annotation reference with pre sorting by name through Samtools Sort Samtools version 0.1.19 within the Htseq count software. Raw counts data obtained from Htseq counts were normalized using DESeq2 Version 1.10.1 in R version 3.2.3 64bit platform using size factors obtained from the total dataset and virtual reference sample based on the data. Genome build: GRCz10 Supplementary files format and content: EXCEL DeSeq2 normalized counts 30 pooled zebrafish wildtype embryos AB background exposed to 1 mM tBOOH. RNA sampled at 48 hpf. Waterborne exposures to either diquat or tBOOH were carried out at three different developmental stages: 2 hpf 48hpf and 96hpf in 3 pools of 30 embryos per condition. Animals were exposed to no treatment 20µM diquat or 1mM tBOOH for a 4 hour dosing period in glass scintillation vials in a 20mL volume of 0.3x Danieau’s. For phenotypic analysis animals were moved to 0.3x Danieau’s for 48 hours post exposure hpe and imaged with a Leica M165 FC stereoscope and DFC310FX camera. For RNA Seq experiments animals were moved to 0.3x Danieau’s for 4 hpe before being flash frozen using liquid nitrogen and stored at 80°C. Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. Adults were held in 2:1 female to male groups at a density of ≤ 5 fish/l in aerated filtered and re circulated system water 28.5 °C and fertilized eggs were obtained from multiple group breedings. Wildtype animals were on the AB background Zebrafish International Resource Center Eugene OR. Fish husbandry protocols were approved by the Bates College Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3320 01 and the University of Michigan Institutional Animal Care and Use Committee Animal Welfare Assurance Number Number A3114 01. To create a germline nfe2 knockout a pair of vectors containing TAL effector nucleases TALENs targeting exon 3 of nfe2 were generated using the REAL Restriction Enzyme And Ligation assembly method. Component plasmids were obtained from Addgene www.addgene.org/talengineering/talenkit/. Briefly target sites were selected and TALENs designed using Zifit http://zifit.partners.org/ZiFiT/ followed by assembly. mRNA was synthesized from the vectors and injected into single cell zebrafish embryos on an AB/TL hybrid background Shavit et al in preparation. The TALEN target sequences are: five prime TCACCCACCTCTTATGAG three prime and five prime CATGACTACACGTGGTCA three prime. A subsequent founder deletion of eight base pairs GCACATGA was found via sequencing in exon three starting at nucleotide position 468 from the translational start site; this deletion resulted in a frame shift causing a change in protein sequence starting at amino acid 111 M→ D and the introduction of a premature stop codon 13 amino acids later. The frameshift was introduced 161 amino acids prior to the Cap'n'collar CNC family basic leucine zipper domain that is responsible for DNA binding. developmental stage:embryo|genotype:wildtype GSM2203891 GSM2203891: 48 hpf AB tBOOH rep 3; Danio rerio; RNA Seq GSM2203891   1 Total RNA was isolated from pooled animals using the Aurum Total RNA Mini Kit BioRad Hercules California USA following the manufacturer’s protocol. At HudsonAlpha Institute for Biotechnology Huntsville AL USA the quality of the RNA was checked using Caliper Instrumentation PerkinElmer Waltham MA USA and RiboGreen reagents Invitrogen Carlsbad CA USA. Directional mRNA libraries with polyA selection were made using New England Biolabs Ipswich MA USA reagents: NEBNext Poly A mRNA Isolation Magnetic Module NEBNext First Strand Synthesis Module NEBNext Second Strand Synthesis Module with dUTP NEBNext End Repair Module NEBNext dA Tailing Module and the NEBNext Quick Ligation Module. Quality analysis of the libraries was carried out using PicoGreen Thermo Fisher Waltham MA USA and Caliper instrumentation PerkinElmer Waltham MA USA. 50 base pair bp paired end libraries were sequenced using the Illumina HiSeq v4 platform at HudsonAlpha Institute for Biotechnology Huntsville AL USA. GEO Accession:GSM2203891 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2000   SRP076704     48_AB_tBOOH_3_fwd.fastq.gz 48_AB_tBOOH_3_rev.fastq.gz fastq fastq 2757349500.0 27573495.0 GSM2203891 r1 0:50 1:50 A:739178336;C:634767141;G:640407032;T:741490318;N:1506673 50 50     739178336 634767141 640407032 741490318 1506673 SRX1852513 SRS1509694 SRA434415 GEO Biochemistry & Biomedical Sciences, McMaster University 2 0.94923 0.95402 0.09987 0.09935 0.68779 0.68852 0.47217 0.47394 50 50 B B biological fallback assumption illumina hiseq_era unknown poly_a nebnext bulk unknown unknown   Canada 2016-06-17 Multi-stage Multi-stage Embryo Imprecise All anatomical structures

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;